Compositions, kits, and methods for identification, assessment, prevention and treatment of pancreatic adenocarcinoma in humans

ABSTRACT

The invention relates to compositions, kits, and methods for detecting, characterizing, preventing, and treating human pancreatic adenocarcinoma. A variety of chromosomal regions (MCRs) and markers in the MCRs, are provided that are correlated with cancer. In particular, chromosomal regions and markers in the MCR 50.06-62.89 Mb of human chromosome 19, are provided, wherein alterations in the copy number of the MCR and/or alterations in the amount, structure, and/or activity of one or more of the markers in the MCR is correlated with the presence of pancreatic adenocarcinoma.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/575,795, which was filed on May 28, 2004 and U.S. ProvisionalApplication No. 60/580,337, which was filed on Jun. 15, 2004, both ofwhich are hereby incorporated by refemce in there entirety.

GOVERNMENT FUNDING

Work described herein was supported, at least in part, by NationalInstitutes of Health (NIH) under grant RO1CA99041, R01CA86379,R01CA84628, and T32 CA09382. The government may therefore have certainrights to this invention.

BACKGROUND OF THE INVENTION

Cancer represents the phenotypic end-point of multiple genetic lesionsthat endow cells with a full range of biological properties required fortumorigenesis. Indeed, a hallmark genomic feature of many cancers,including, for example, pancreatic cancer, breast cancer, ovariancancer, and colon cancer, is the presence of numerous complex chromosomestructural aberrations—including non-reciprocal translocations,amplifications and deletions.

Karyotype analyses (Johansson, B., et al. (1992) Cancer 69, 1674-81;Bardi, G., et al. (1993) Br J Cancer 67, 1106-12; Griffin, C. A., et al.(1994) Genes Chromosomes Cancer 9, 93-100; Griffin, C. A., et al. (1995)Cancer Res 55, 2394-9; Gorunova, L., et al. (1995) Genes ChromosomesCancer 14, 259-66; Gorunova, L., et al. (1998) Genes Chromosomes Cancer23, 81-99), chromosomal CGH and array CGH (Wolf M et al. (2004)Neoplasia 6 (3) 240; Kimura Y, et al. (2004) Mod. Pathol. 21 May (epub);Pinkel, et al. (1998) Nature Genetics 20:211; Solinas-Toldo, S., et al.(1996) Cancer Res 56, 3803-7; Mahlamaki, E. H., et al. (1997) GenesChromosomes Cancer 20, 383-91; Mahlamaki, E. H., et al. (2002) GenesChromosomes Cancer 35, 353-8; Fukushige, S., et al. (1997) GenesChromosomes Cancer 19:161-9; Curtis, L. J., et al. (1998) Genomics 53,42-55; Ghadimi, B. M., et al. (1999) Am J Pathol 154, 525-36; Armengol,G., et al. (2000) Cancer Genet Cytogenet 116, 133-41), fluorescence insitu hybridization (FISH) analysis (Nilsson M et al. (2004) Int J Cancer109(3):363-9; Kawasaki K et al. (2003) Int J Mol. Med. 12(5):727-31) andloss of heterozygosity (LOH) mapping (Wang Z C et al. (2004) Cancer Res64(1):64-71; Seymour, A. B., et al. (1994) Cancer Res 54, 2761-4; Hahn,S. A., et al. (1995) Cancer Res 55, 4670-5; Kimura, M., et al. (1996)Genes Chromosomes Cancer 17, 88-93) have identified recurrent regions ofcopy number change or allelic loss in various cancers. For example, inpancreatic cancer, frequent gains have been mapped to 3q, 5p, 7p, 8q,11q, 12p, 17q and 20q and losses to 3p, 4q, 6q, 8p, 9p, 10q, 12q, 13q,17p, 18q and 21q and 22q. In some instances, validated oncogenes andtumor suppressor genes residing within these loci have been identified,including MYC (8q24), p^(INK4A) (9p21), p53 (17p13), SMAD4 (18q21) andAKT2 (19q13). However, for the majority of amplified and deleted lociand resident genes, the presumed cancer-relevant targets remain to bediscovered.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the identificationof specific regions of the genome (referred to herein as minimal commonregions (MCRs)), of recurrent copy number change which are containedwithin certain chromosomal regions (loci) and are associated withcancer. These MCRs were identified using a novel cDNA or oligomer-basedplatform and bioinformatics tools which allowed for the high-resolutioncharacterization of copy-number alterations in the pancreaticadenocarcinoma genome (see Example 1). The present invention is based,also in part, on the identification of markers residing within the MCRsof the invention, which are also associated with cancer.

Accordingly, in one aspect, the present invention provides methods ofassessing whether a subject is afflicted with cancer or at risk fordeveloping cancer, comprising comparing the copy number of an MCR in asubject sample to the normal copy number of the MCR, wherein the MCR isselected from the group consisting of the MCRs listed in Table 1, andwherein an altered copy number of the MCR in the sample indicates thatthe subject is afflicted with cancer or at risk for developing cancer.In one embodiment, the copy number is assessed by fluorescent in situhybridization (FISH). In another embodiment, the copy number is assessedby quantitative PCR (qPCR). In still another embodiment, the normal copynumber is obtained from a control sample. In yet another embodiment, thesample is selected from the group consisting of tissue, whole blood,serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool,bile, pancreatic juice, and pancreatic tissue.

In another aspect, the invention provides methods of assessing whether asubject is afflicted with cancer or at risk for developing cancercomprising comparing the amount, structure, and/or activity of a markerin a subject sample, wherein the marker is a marker which resides in anMCR listed in Table 1, and the normal amount, structure, and/or activityof the marker, wherein a significant difference between the amount,structure, and/or activity of the marker in the sample and the normalamount, structure, and/or activity is an indication that the subject isafflicted with cancer or at risk for developing cancer. In oneembodiment, the marker is selected from the group consisting of themarkers listed in Table 4 or Table 5. In another embodiment, the amountof the marker is determined by determining the level of expression ofthe marker. In yet another embodiment, the level of expression of themarker in the sample is assessed by detecting the presence in the sampleof a protein corresponding to the marker. The presence of the proteinmay be detected using a reagent which specifically binds with theprotein. In one embodiment, the reagent is selected from the groupconsisting of an antibody, an antibody derivative, and an antibodyfragment. In another embodiment, the level of expression of the markerin the sample is assessed by detecting the presence in the sample of atranscribed polynucleotide or portion thereof, wherein the transcribedpolynucleotide comprises the marker. In one embodiment, the transcribedpolynucleotide is an mRNA or cDNA. The level of expression of the markerin the sample may also be assessed by detecting the presence in thesample of a transcribed polynucleotide which anneals with the marker oranneals with a portion of a polynucleotide wherein the polynucleotidecomprises the marker, under stringent hybridization conditions.

In another embodiment, the amount of the marker is determined bydetermining copy number of the marker. The copy number of the MCRs ormarkers may be assessed by comparative genomic hybridization (CGH),e.g., array CGH. In still another embodiment, the normal amount,structure, and/or activity is obtained from a control sample. In yetanother embodiment, the sample is selected from the group consisting oftissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinalfluid, urine, stool, bile, pancreatic juice, and pancreatic tissue.

In another aspect, the invention provides methods for monitoring theprogression of cancer in a subject comprising a) detecting in a subjectsample at a first point in time, the amount and/or activity of a marker,wherein the marker is a marker which resides in an MCR listed in Table1; b) repeating step a) at a subsequent point in time; and c) comparingthe amount and/or activity detected in steps a) and b), and therefrommonitoring the progression of cancer in the subject. In one embodiment,the marker is selected from the group consisting of the markers listedin Table 4 or Table 5. In another embodiment, the sample is selectedfrom the group consisting of tissue, whole blood, serum, plasma, buccalscrape, saliva, cerebrospinal fluid, urine, stool, bile, pancreaticjuice, and pancreatic tissue. In still another embodiment, the samplecomprises cells obtained from the subject. In yet another embodiment,between the first point in time and the subsequent point in time, thesubject has undergone treatment for cancer, has completed treatment forcancer, and/or is in remission.

In still another aspect, the invention provides methods of assessing theefficacy of a test compound for inhibiting cancer in a subjectcomprising comparing the amount and/or activity of a marker in a firstsample obtained from the subject and maintained in the presence of thetest compound, wherein the marker is a marker which resides in an MCRlisted in Table 1, and the amount and/or activity of the marker in asecond sample obtained from the subject and maintained in the absence ofthe test compound, wherein a significantly higher amount and/or activityof a marker in the first sample which is deleted in cancer, relative tothe second sample, is an indication that the test compound isefficacious for inhibiting cancer, and wherein a significantly loweramount and/or activity of the marker in the first sample which isamplified in cancer, relative to the second sample, is an indicationthat the test compound is efficacious for inhibiting cancer in thesubject. In one embodiment, the first and second samples are portions ofa single sample obtained from the subject. In another embodiment, thefirst and second samples are portions of pooled samples obtained fromthe subject. In one embodiment, the marker is selected from the groupconsisting of the markers listed in Table 4 or Table 5.

In yet another aspect, the invention provides methods of assessing theefficacy of a therapy for inhibiting cancer in a subject comprisingcomparing the amount and/or activity of a marker in the first sampleobtained from the subject prior to providing at least a portion of thetherapy to the subject, wherein the marker is a marker which resides inan MCR listed in Table 1, and the amount and/or activity of the markerin a second sample obtained from the subject following provision of theportion of the therapy, wherein a significantly higher amount and/oractivity of a marker in the first sample which is deleted in cancer,relative to the second sample, is an indication that the test compoundis efficacious for inhibiting cancer and wherein a significantly loweramount and/or activity of a marker in the first sample which isamplified in cancer, relative to the second sample, is an indicationthat the therapy is efficacious for inhibiting cancer in the subject. Inone embodiment, the marker is selected from the group consisting of themarkers listed in Table 4 or Table 5.

Another aspect of the invention provides methods of selecting acomposition capable of modulating cancer comprising obtaining a samplecomprising cancer cells; contacting said cells with a test compound; anddetermining the ability of the test compound to modulate the amountand/or activity of a marker, wherein the marker is a marker whichresides in an MCR listed in Table 1, thereby identifying a modulator ofcancer. In one embodiment, the marker is selected from the groupconsisting of the markers listed in Table 4 or Table 5. The cells may beisolated from, e.g., an animal model of cancer, a cancer cell line,e.g., a pancreatic cancer cell line originating from a pancreatic tumor,or from a subject suffering from cancer.

Yet another aspect of the invention provides methods of selecting acomposition capable of modulating cancer comprising contacting a markerwith a test compound; and determining the ability of the test compoundto modulate the amount and/or activity of a marker, wherein the markeris a marker which resides in an MCR listed in Table 1, therebyidentifying a composition capable of modulating cancer. In oneembodiment, the marker is selected from the group consisting of themarkers listed in Table 4 or Table 5. In another embodiment, the methodfurther comprises administering the test compound to an animal model ofcancer. In still another embodiment, the modulator inhibits the amountand/or activity of a gene or protein corresponding to a marker set forthin Table 1 which is amplified, e.g., a marker selected from the markerslisted in Table 5. In yet another embodiment, the modulator increasesthe amount and/or activity of a gene or protein corresponding to amarker set forth in Table 1 which is deleted, e.g., a marker selectedfrom the markers listed in Table 4.

In another aspect, the invention provides kits for assessing the abilityof a compound to inhibit cancer comprising a reagent for assessing theamount, structure, and/or activity of a marker, wherein the marker is amarker which resides in an MCR listed in Table 1. In one embodiment, themarker selected from the group consisting of the markers listed in Table4 or Table 5.

The invention also provides kits for assessing whether a subject isafflicted with cancer comprising a reagent for assessing the copy numberof an MCR selected from the group consisting of the MCRs listed in Table1, as well as kits for assessing whether a subject is afflicted withcancer, the kit comprising a reagent for assessing the amount,structure, and/or activity of a marker. In one embodiment, the markerselected from the group consisting of the markers listed in Table 4 orTable 5.

In another aspect, the invention provides kits for assessing thepresence of human cancer cells comprising an antibody or fragmentthereof, wherein the antibody or fragment thereof specifically bindswith a protein corresponding to a marker, wherein the marker is a markerwhich resides in an MCR listed in Table 1. In one embodiment, the markerselected from the group consisting of the markers listed in Table 4 orTable 5.

In still another aspect, the invention provides kits for assessing thepresence of cancer cells comprising a nucleic acid probe wherein theprobe specifically binds with a transcribed polynucleotide correspondingto a marker, wherein the marker is a marker which resides in an MCRlisted in Table 1. In one embodiment, the marker selected from the groupconsisting of the markers listed in Table 4 or Table 5.

In yet another aspect, the invention provides methods of treating asubject afflicted with cancer comprising administering to the subject amodulator of the amount and/or activity of a gene or proteincorresponding to a marker, wherein the marker is a marker which residesin an MCR listed in Table 1. In one embodiment, the marker selected fromthe group consisting of the markers listed in Table 4 or Table 5.

The invention also provides methods of treating a subject afflicted withcancer comprising administering to the subject a compound which inhibitsthe amount and/or activity of a gene or protein corresponding to amarker which resides in an MCR listed in Table 1 which is amplified incancer, e.g., a marker selected from the markers listed in Table 5,thereby treating a subject afflicted with cancer. In one embodiment, thecompound is administered in a pharmaceutically acceptable formulation.In another embodiment, the compound is an antibody or an antigen bindingfragment thereof, which specifically binds to a protein corresponding tothe marker. For example, the antibody may be conjugated to a toxin or achemotherapeutic agent. In still another embodiment, the compound is anRNA interfering agent, e.g., an siRNA molecule or an shRNA molecule,which inhibits expression of a gene corresponding to the marker. In yetanother embodiment, the compound is an antisense oligonucleotidecomplementary to a gene corresponding to the marker. In still anotherembodiment, the compound is a peptide or peptidomimetic, a smallmolecule which inhibits activity of the marker, e.g., a small moleculewhich inhibits a protein-protein interaction between a marker and atarget protein, or an aptamer which inhibits expression or activity ofthe marker.

In another aspect, the invention provides methods of treating a subjectafflicted with cancer comprising administering to the subject a compoundwhich increases expression or activity of a gene or proteincorresponding to a marker which resides in an MCR listed in Table 1which is deleted in cancer, e.g., a marker selected from the markerslisted in Table 4, thereby treating a subject afflicted with cancer. Inone embodiment, the compound is a small molecule.

The invention also includes methods of treating a subject afflicted withcancer comprising administering to the subject a protein correspondingto a marker, e.g., a marker selected from the markers listed in Table 4,thereby treating a subject afflicted with cancer. In one embodiment, theprotein is provided to the cells of the subject, by a vector comprisinga polynucleotide encoding the protein. In still another embodiment, thecompound is administered in a pharmaceutically acceptable formulation.

The present invention also provides isolated proteins, or fragmentsthereof, corresponding to a marker selected from the markers listed inTable 4 or Table 5.

In another aspect, the invention provides isolated nucleic acidmolecules, or fragments thereof, corresponding to a marker selected fromthe markers listed in Table 4 or Table 5.

In still another aspect, the invention provides isolated antibodies, orfragments thereof, which specifically bind to a protein corresponding toa marker selected from the markers listed in Table 4 or Table 5.

In yet another aspect, the invention provides an isolated nucleic acidmolecule, or fragment thereof, contained within an MCR selected from theMCRs listed in Table 1, wherein said nucleic acid molecule has analtered amount, structure, and/or activity in cancer. The invention alsoprovides an isolated polypeptide encoded by the nucleic acid molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict the genomic profiles from pancreatic adenocarcinomasamples. Array-CGH profiles with x-axis coordinates representing cDNAprobes ordered by genomic map positions. Segmented data is displayed inred, median filtered (3 nearest neighbors) in blue and raw data inblack. (1A) Whole-genome profiles of primary tumor specimen PA.T.7692(top) and cell line Panc 10.05 (bottom). Note the presence of focalhigh-level amplifications and deletions as well as large regional gainsand losses in both samples. (1B) Recurrence of chromosomal alterations.Top: Integer-value recurrence of CNAs in segmented data (Y-axis) plottedfor each cDNA probe evenly aligned along the x-axis in genome order.Dark red or green bars denote gain or loss of chromosome material.Bright red or green bars represent probes within regions ofamplification or deletion. Bottom: TreeView showing discrete CNAs withinall samples. Red represents chromosomal gain and green denoteschromosomal loss. (1C) CGH profiles of 12p12.3-q13.3 locus (Locus # 15of Table 1) in three samples illustrating the definition of the physicalextent, peak profile and MCRs for that locus. Note that the left MCR isdefined by the overlap between samples on top and bottom, while theright MCR is defined by the overlap between the two samples on top.Since data points are plotted on the x-axis by genomic map positions,gaps in the profiles encompass regions of copy number transition forwhich there is no data point.

FIGS. 2A-2B depict that QPCR verifies complexity within CNAs. (2A)Chromosome 7 CGH profiles (Left panel) showing amplification of adiscrete region of 7q22 in both the AsPC-1 cell line and PA.T.14172(Locus # 9, Table 1), with MCR defined by both samples (outlined bydashed lines). Letters A-D indicate the relative positions of QPCRassays (Right panel), which confirm the gene copy alterations in AsPC-1(dark gray bars) and PA.T.14172 (light gray bars). (2B) Chromosome 9array-CGH profile (Left panel) for a complex CNA in the HUP-T3 cellline. Homozygous deletion of the known target p16^(1nk4a) is confirmedby QPCR (Right panel), which also verifies the existence of twodiscrete, focal amplicons and a narrow region of one-copy loss revealedby array-CGH. Note that CNAs covered by only one or two probes are notidentified by the segmentation algorithm.

FIGS. 3A-3C depict that combined array-CGH and expression analysisfacilitates identification of candidate genes. (3A) Analysis of17q23.2-25.3 locus (Locus #21, Table 1) in cell line Hup T3. Top panel:array-CGH profile of HUP-T3. Bottom panel: Expression profile of geneson Affymetrix U133A array within the specified locus for the HUP-T3 cellline. The subset of genes exhibiting prominent gene-dosage correlatedexpression fall within the peak of the locus (arrows). (3B) Analysis of9p24.3-21.2 locus (Locus #41, Table 1) in the cell line BxPC-3. Toppanel: array-CGH profile of 9p region. Bottom panel: Affymetrix™expression profile of genes mapping to the same region. Note thedramatically reduced expression of the p16^(INK4A) gene (arrows) withinthe MCR. (3C) Correlation of p16^(INK4A) expression and copy number in24 cell lines was analyzed. Note the bimodal distribution of bothexpression values and copy number values for this gene across allsamples (green lines). The box outlined in dots defines those samples(BxPC-3, MiaPaCa, Capan 1, Hup-T3 and Dan-G) in which p16^(INK4A) ishomozygously deleted and not expressed. The box outlined in solid linesencloses samples (Panc-1, Panc 03.27, SW1990, Panc 08.13, Hup-T4 andPanc 02.13) in which p16^(INK4A) is present but with absent or reducedexpression.

FIG. 4 depicts a histogram of segmented profiles showing the peak at aLog₂ ratio of 0 as a result of mode centering. Outer lines mark 3% (del)and 97% (amp) quantiles; inner lines mark+/−4 standard deviation ofmiddle 50% of data.

FIG. 5 depicts a comparison of array-CGH profiles of pancreaticadenocarcinoma primary tumors and cell lines. A pseudo-karyotypepresentation of an integer-value recurrence plot by chromosome is shownhere for both primary tumors (PT) and cell lines (CL). Gain is shown onthe right of the chromosome and loss on the left of the chromosome.Important regions of similarity between PT and CL are highlighted withboxes and chromosomes with prominent discrepancies between PT and CL arecircled.

FIG. 6 depicts a whole-genome correlation of copy number and expressionin the cell line Capan-1. Median filtered (width=31 probes) array-CGHand expression data are indicated in light blue and gold, respectively.Note that fluctuations in average gene expression correlate with changesin chromosome copy number (R=0.66).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the identificationof specific regions of the genome (referred to herein as minimal commonregions (MCRs)), of recurrent copy number change which are containedwithin certain chromosomal regions (loci) and are associated withcancer. These MCRs were identified using a novel cDNA or oligomer-basedplatform and bioinformatics tools which allowed for the high-resolutioncharacterization of copy-number alterations in the pancreaticadenocarcinoma genome (see Example 1).

To arrive at the identified loci and MCRs, array comparative genomichybridization (array-CGH) was utilized to define copy number aberrations(CNAs) (gains and losses of chromosomal regions) in pancreaticadenocarcinoma cell lines and tumor specimens.

Segmentation analysis of the raw profiles to filter noise from thedataset (as described by Olshen and Venkatraman, Olshen, A. B., andVenkatraman, E. S. (2002) ASA Proceedings of the Joint StatisticalMeetings 2530-2535; Ginzinger, D. G. (2002) Exp Hematol 30, 503-12;Golub, T. R., et al. (1999) Science 286, 531-7; Hyman, E., et al. (2002)Cancer Res 62, 6240-5; Lucito, R., et al. (2003) Genome Res 13,2291-305) was performed and used to identify statistically significantchangepoints in the data.

Identification of loci was based on an automated computer algorithm thatutilized several basic criteria as follows: 1) segments above or belowcertain percentiles were identified as altered; 2) if two or morealtered segments were adjacent in a single profile separated by lessthan 500KB, the entire region spanned by the segments was considered tobe an altered span; 3) highly altered segments or spans that wereshorter than 20 MB were retained as “informative spans” for definingdiscrete locus boundaries. Longer regions were not discarded, but werenot included in defining locus boundaries; 4) informative to spans werecompared across samples to identify overlapping groups of positive-valueor negative-value segments; each group defines a locus; and 5) MCRs weredefined as contiguous spans having at least 75% of the peak recurrenceas calculated by counting the occurrence of highly altered segments. Iftwo MCRs were separated by a gap of only one probe position, they werejoined. If there were more than 3 MCRs in a locus, the whole region wasreported as a single complex MCR.

A locus-identification algorithm was used that defines informative CNAson the basis of size and achievement of a high significance thresholdfor the amplitude of change. Overlapping CNAs from multiple profileswere then merged in an automated fashion to define a discrete “locus” ofregional copy number change, the bounds of which represent the combinedphysical extend to these overlapping CNAs (FIG. 1C). Each locus wascharacterized by a peak profile, the width and amplitude of whichreflect the contour of the most prominent amplification or deletion forthat locus. Furthermore, within each locus, one or more minimal commonregions (MCRs) were identified across multiple tumor samples (FIG. 1C),with each MCR potentially harboring a distinct cancer-relevant genetargeted for copy number alteration across the sample set.

The locus-identification algorithm defined discrete MCRs within thedataset which were annotated in terms of recurrence, amplitude of changeand representation in both cell lines and primary tumors. These discreteMCRs were prioritized based on four criteria that emphasize recurrenthigh-threshold changes in both primary tumors and cell lines (seeExample 1). Implementation of this prioritization scheme yielded 64 MCRsof the present invention within 54 independent loci, that satisfied atleast three of the four criteria (see Table 1).

The confidence-level ascribed to these prioritized loci was furthervalidated by real-time quantitative PCR (QPCR), which demonstrated 100%concordance with 16 selected MCRs defined by array-CGH. When the MCRs inTable 1 were combined with an additional 81 MCRs (within 66 distinctloci) satisfying 2 out of 4 criteria, this genomic characterization hasproduced a set of 145 MCRs within 121 independent loci (Table 3).

The MCRs identified herein possess a median size of 2.7 Mb, with 21(33%) MCRs spanning 1 Mb or less (median of 0.33 Mb) and possess anaverage of 15 annotated genes. Table 1 lists the cytogenetic bands foreach of the 54 independent loci as well as the locus boundary (Mb) andlocus peak profile. The positions of each of the identified MCRs arealso listed in Table 1, as well as the size and recurrence for each. Forexample, locus #3 represents a chromosomal region of 5q31.1-q31.1 andhas a locus boundary of 133.51-134.33. This locus contains an MCR atposition 133.53-133.56.

Also in Table 1, the loci and MCRs are indicated as having either “gainand amplification” or “loss and deletion,” indicating that each locusand MCR has either (1) increased copy number and/or expression or (2)decreased copy number and/or expression, or deletion, in cancer.Furthermore, genes known to play important roles in the pathogenesis ofpancreatic adenocarcinoma (the p16^(INK4A) and TP53 tumor suppressorsand the MYC, KRAS2 and AKT2 oncogenes) are present within the loci andare also set forth in Table 1.

Complementary expression profile analysis of a significant fraction ofthe genes residing within the MCRs of the present invention provided asubset of markers with statistically significant association betweengene dosage and mRNA expression. Table 4 lists the markers of theinvention which reside in MCRs of deletion and which consequentlydisplay decreased expression by comparison across pancreatic cancer celllines. Table 5 lists the markers of the invention which reside in MCRsof amplification that are overexpressed by comparison, across pancreaticcancer cell lines. Additional markers within the MCRs that have not yetbeen annotated may also be used as markers for cancer as describedherein, and are included in the invention.

The novel methods for identifying chromosomal regions of altered copynumber, as described herein, may be applied to various data sets forvarious diseases, including, but not limited to, cancer. Other methodsmay be used to determine copy number aberrations as are known in theart, including, but not limited to oligonucleotide-based microarrays(Brennan, et al. (2004) In Press; Lucito, et al. (2003) Genome Res.13:2291-2305; Bignell et al. (2004) Genome Res. 14:287-295; Zhao, et al(2004) Cancer Research, In Press), and other methods as described hereinincluding, for example, hybridization methods (FISH).

The amplification or deletion of the MCRs identified herein correlatewith the presence of cancer, e.g., pancreatic cancer and otherepithelial cancers. Furthermore, analysis of copy number and/orexpression levels of the genes residing within each MCR has led to theidentification of individual markers and combinations of markersdescribed herein, the increased and decreased expression and/orincreased and decreased copy number of which correlate with the presenceof cancer, e.g., pancreatic cancer, e.g., in a subject.

Accordingly, methods are provided herein for detecting the presence ofcancer in a sample, the absence of cancer in a sample, and othercharacteristics of cancer that are relevant to prevention, diagnosis,characterization, and therapy of cancer in a subject by evaluatingalterations in the amount, structure, and/or activity of a marker. Forexample, evaluation of the presence, absence or copy number of the MCRsidentified herein, or by evaluating the copy number, expression level,protein level, protein activity, presence of mutations (e.g.,substitution, deletion, or addition mutations) which affect activity ofthe marker, or methylation status of any one or more of the markerswithin the MCRs (e.g., the markers set forth in Tables 4 and 5), iswithin the scope of the invention.

Methods are also provided herein for the identification of compoundswhich are capable of inhibiting cancer, in a subject, and for thetreatment, prevention, and/or inhibition of cancer using a modulator,e.g., an agonist or antagonist, of a gene or protein marker of theinvention.

Although the MCRs and markers described herein were identified inpancreatic cancer samples, the methods of the invention are in no waylimited to use for the prevention, diagnosis, characterization, therapyand prevention of pancreatic cancer, e.g., the methods of the inventionmay be applied to any cancer, as described herein.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “tumor” or “cancer” refer to the presence of cells possessingcharacteristics typical of cancer-causing cells, such as uncontrolledproliferation, immortality, metastatic potential, rapid growth andproliferation rate, and certain characteristic morphological features.Cancer cells are often in the form of a tumor, but such cells may existalone within an animal, or may be a non-tumorigenic cancer cell, such asa leukemia cell. As used herein, the term “cancer” includes premalignantas well as malignant cancers. Cancers include, but are not limited to,pancreatic cancer, e.g., pancreatic adenocarcinoma, melanomas, breastcancer, lung cancer, bronchus cancer, colorectal cancer, prostatecancer, pancreas cancer, stomach cancer, ovarian cancer, urinary bladdercancer, brain or central nervous system cancer, peripheral nervoussystem cancer, esophageal cancer, cervical cancer, uterine orendometrial cancer, cancer of the oral cavity or pharynx, liver cancer,kidney cancer, testicular cancer, biliary tract cancer, small bowel orappendix cancer, salivary gland cancer, thyroid gland cancer, adrenalgland cancer, osteosarcoma, chondrosarcoma, cancer of hematologicaltissues, and the like.

The term “pancreatic cancer” or “neoplasia” as used herein, includesPanIns, adenomas, adenocarcinomas, gastrinomas, somatostatinomas,insulinomas and glucagonomas of the pancreas.

As used herein, the term “adenocarcinoma” is carcinoma that develops inthe lining or inner surface of an organ and is derived from glandulartissue or in which the tumor cells form recognizable glandularstructures.

As used interchangeably herein, the terms, “pancreatic adenocarcinoma,”or “pancreatic ductal adenocarcinoma” is an adenocarcinoma of thepancreas. In one embodiment, pancreatic adenocarcinomas arise from theprogression of premalignant lesions that occur in the pancreatic ducts(pancreatic intraepithelial neoplasia, referred to herein as “PanIN”).The methods described herein may be used to detect premalignant cancers,e.g., PanIns, as well as malignant cancers.

A “minimal common region (MCR),” as used herein, refers to a contiguouschromosomal region which displays either gain and amplification(increased copy number) or loss and deletion (decreased copy number) inthe genome of a cancer. An MCR includes at least one nucleic acidsequence which has increased or decreased copy number and which isassociated with a cancer. The MCRs of the instant invention include, butare not limited to, those set forth in Table 1.

A “marker” is a gene or protein which may be altered, wherein saidalteration is associated with cancer. The alteration may be in amount,structure, and/or activity in a cancer tissue or cancer cell, ascompared to its amount, structure, and/or activity, in a normal orhealthy tissue or cell (e.g., a control), and is associated with adisease state, such as cancer. For example, a marker of the inventionwhich is associated with cancer may have altered copy number, expressionlevel, protein level, protein activity, or methylation status, in acancer tissue or cancer cell as compared to a normal, healthy tissue orcell. Furthermore, a “marker” includes a molecule whose structure isaltered, e.g., mutated (contains an allelic variant), e.g., differs fromthe wild type sequence at the nucleotide or amino acid level, e.g., bysubstitution, deletion, or addition, when present in a tissue or cellassociated with a disease state, such as cancer.

The term “altered amount” of a marker or “altered level” of a markerrefers to increased or decreased copy number of a marker or chromosomalregion, e.g., MCR, and/or increased or decreased expression level of aparticular marker gene or genes in a cancer sample, as compared to theexpression level or copy number of the marker in a control sample. Theterm “altered amount” of a marker also includes an increased ordecreased protein level of a marker in a sample, e.g., a cancer sample,as compared to the protein level of the marker in a normal, controlsample. Furthermore, an altered amount of a marker may be determined bydetecting the methylation status of a marker, as described herein, whichmay affect the expression or activity of a marker.

The amount of a marker, e.g., expression or copy number of a marker orMCR, or protein level of a marker, in a subject is “significantly”higher or lower than the normal amount of a marker or MCR, if the amountof the marker is greater or less, respectively, than the normal level byan amount greater than the standard error of the assay employed toassess amount, and preferably at least twice, and more preferably three,four, five, ten or more times that amount. Alternately, the amount ofthe marker or MCR in the subject can be considered “significantly”higher or lower than the normal amount if the amount is at least abouttwo, and preferably at least about three, four, or five times, higher orlower, respectively, than the normal amount of the marker or MCR.

The “copy number of a gene” or the “copy number of a marker” refers tothe number of DNA sequences in a cell encoding a particular geneproduct. Generally, for a given gene, a mammal has two copies of eachgene. The copy number can be increased, however, by gene amplificationor duplication, or reduced by deletion.

The “normal” copy number of a marker or MCR or “normal” level ofexpression of a marker is the level of expression, copy number of themarker, or copy number of the MCR, in a biological sample, e.g., asample containing tissue, whole blood, serum, plasma, buccal scrape,saliva, cerebrospinal fluid, urine, stool, bile, pancreatic juice, andpancreatic tissue, from a subject, e.g. a human, not afflicted withcancer.

The term “altered level of expression” of a marker or MCR refers to anexpression level or copy number of a marker in a test sample e.g., asample derived from a patient suffering from cancer, that is greater orless than the standard error of the assay employed to assess expressionor copy number, and is preferably at least twice, and more preferablythree, four, five or ten or more times the expression level or copynumber of the marker or MCR in a control sample (e.g., sample from ahealthy subjects not having the associated disease) and preferably, theaverage expression level or copy number of the marker or MCR in severalcontrol samples. The altered level of expression is greater or less thanthe standard error of the assay employed to assess expression or copynumber, and is preferably at least twice, and more preferably three,four, five or ten or more times the expression level or copy number ofthe marker or MCR in a control sample (e.g., sample from a healthysubjects not having the associated disease) and preferably, the averageexpression level or copy number of the marker or MCR in several controlsamples.

An “overexpression” or “significantly higher level of expression or copynumber” of a marker or MCR refers to an expression level or copy numberin a test sample that is greater than the standard error of the assayemployed to assess expression or copy number, and is preferably at leasttwice, and more preferably three, four, five or ten or more times theexpression level or copy number of the marker or MCR in a control sample(e.g., sample from a healthy subject not afflicted with cancer) andpreferably, the average expression level or copy number of the marker orMCR in several control samples.

“Methylation status” of a marker refers to the methylation pattern,e.g., methylation of the promoter of the marker, and/or methylationlevels of the marker. DNA methylation is a heritable, reversible andepigenetic change. Yet, DNA methylation has the potential to alter geneexpression, which has developmental and genetic consequences. DNAmethylation has been linked to cancer, as described in, for example,Laird, et al. (1994) Human Molecular Genetics 3:1487-1495 and Laird, P.(2003) Nature 3:253-266, the contents of which are incorporated hereinby reference. For example, methylation of CpG oligonucleotides in thepromoters of tumor suppressor genes can lead to their inactivation. Inaddition, alterations in the normal methylation process are associatedwith genomic instability (Lengauer et al. Proc. Natl. Acad. Sci. USA94:2545-2550, 1997). Such abnormal epigenetic changes may be found inmany types of cancer and can, therefore, serve as potential markers foroncogenic transformation.

Methods for determining methylation include restriction landmark genomicscanning (Kawai et al., Mol. Cell. Biol. 14:7421-7427, 1994),methylation-sensitive arbitrarily primed PCR (Gonzalgo et al., CancerRes. 57:594-599, 1997); digestion of genomic DNA withmethylation-sensitive restriction enzymes followed by Southern analysisof the regions of interest (digestion-Southern method); PCR-basedprocess that involves digestion of genomic DNA withmethylation-sensitive restriction enzymes prior to PCR amplification(Singer-Sam et al., Nucl. Acids Res. 18:687, 1990); genomic sequencingusing bisulfite treatment (Frommer et al., Proc. Natl. Acad. Sci. USA89:1827-1831, 1992); methylation-specific PCR (MSP) (Herman et al. Proc.Natl. Acad. Sci. USA 93:9821-9826, 1992); and restriction enzymedigestion of PCR products amplified from bisulfite-converted DNA (Sadriand Hornsby, Nucl. Acids Res. 24:5058-5059, 1996; and Xiong and Laird,Nucl. Acids. Res. 25:2532-2534, 1997); PCR techniques for detection ofgene mutations (Kuppuswamyet et al., Proc. Natl. Acad. Sci. USA88:1143-1147, 1991) and quantitation of allelic-specific expression(Szabo and Mann, Genes Dev. 9:3097-3108, 1995; and Singer-Sam et al.,PCR Methods Appl. 1:160-163, 1992); and methods described in U.S. Pat.No. 6,251,594, the contents of which are incorporated herein byreference. An integrated genomic and epigenomic analysis as described inZardo, et al. (2000) Nature Genetics 32:453-458, may also be used.

The term “altered activity” of a marker refers to an activity of amarker which is increased or decreased in a disease state, e.g., in acancer sample, as compared to the activity of the marker in a normal,control sample. Altered activity of a marker may be the result of, forexample, altered expression of the marker, altered protein level of themarker, altered structure of the marker, or, e.g., an alteredinteraction with other proteins involved in the same or differentpathway as the marker or altered interaction with transcriptionalactivators or inhibitors, or altered methylation status.

The term “altered structure” of a marker refers to the presence ofmutations or allelic variants within the marker gene or maker protein,e.g., mutations which affect expression or activity of the marker, ascompared to the normal or wild-type gene or protein. For example,mutations include, but are not limited to substitutions, deletions, oraddition mutations. Mutations may be present in the coding or non-codingregion of the marker.

A “marker nucleic acid” is a nucleic acid (e.g., DNA, mRNA, cDNA)encoded by or corresponding to a marker of the invention. For example,such marker nucleic acid molecules include DNA (e.g., cDNA) comprisingthe entire or a partial sequence of any of the nucleic acid sequencesset forth in Tables 4 or 5 or the complement or hybridizing fragment ofsuch a sequence. The marker nucleic acid molecules also include RNAcomprising the entire or a partial sequence of any of the nucleic acidsequences set forth in Tables 4 or 5 or the complement of such asequence, wherein all thymidine residues are replaced with uridineresidues. A “marker protein” is a protein encoded by or corresponding toa marker of the invention. A marker protein comprises the entire or apartial sequence of a protein encoded by any of the sequences set forthin Tables 4 or 5 or a fragment thereof. The terms “protein” and“polypeptide” are used interchangeably herein.

A “marker,” as used herein, includes any nucleic acid sequence presentin an MCR as set forth in Table 1, or a protein encoded by such asequence.

Markers identified herein include diagnostic and therapeutic markers. Asingle marker may be a diagnostic marker, a therapeutic marker, or botha diagnostic and therapeutic marker.

As used herein, the term “therapeutic marker” includes markers, e.g.,markers set forth in Tables 4 and 5, which are believed to be involvedin the development (including maintenance, progression, angiogenesis,and/or metastasis) of cancer. The cancer-related functions of atherapeutic marker may be confirmed by, e.g., (1) increased or decreasedcopy number (by, e.g., fluorescence in situ hybridization (FISH) orquantitative PCR (qPCR)) or mutation (e.g., by sequencing),overexpression or underexpression (e.g., by in situ hybridization (ISH),Northern Blot, or qPCR), increased or decreased protein levels (e.g., byimmunohistochemistry (IHC)), or increased or decreased protein activity(determined by, for example, modulation of a pathway in which the markeris involved), e.g., in more than about 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 20%, 25%, or more of human cancers; (2) theinhibition of cancer cell proliferation and growth, e.g., in soft agar,by, e.g., RNA interference (“RNAi”) of the marker; (3) the ability ofthe marker to enhance transformation of mouse embryo fibroblasts (MEFs)by oncogenes, e.g., Myc and RAS, or by RAS alone; (4) the ability of themarker to enhance or decrease the growth of tumor cell lines, e.g., insoft agar; (5) the ability of the marker to transform primary mousecells in SCID explant; and/or; (6) the prevention of maintenance orformation of tumors, e.g., tumors arising de novo in an animal or tumorsderived from human cancer cell lines, by inhibiting or activating themarker. In one embodiment, a therapeutic marker may be used as adiagnostic marker.

As used herein, the term “diagnostic marker” includes markers, e.g.,markers set forth in Tables 4 and 5, which are useful in the diagnosisof cancer, e.g., over- or under-activity emergence, expression, growth,remission, recurrence or resistance of tumors before, during or aftertherapy. The predictive functions of the marker may be confirmed by,e.g., (1) increased or decreased copy number (e.g., by FISH or qPCR),overexpression or underexpression (e.g., by ISH, Northern Blot, orqPCR), increased or decreased protein level (e.g., by IHC), or increasedor decreased activity (determined by, for example, modulation of apathway in which the marker is involved), e.g., in more than about 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, or more of humancancers; (2) its presence or absence in a biological sample, e.g., asample containing tissue, whole blood, serum, plasma, buccal scrape,saliva, cerebrospinal fluid, urine, stool, bile, pancreatic juice, andpancreatic tissue from a subject, e.g. a human, afflicted with cancer;(3) its presence or absence in clinical subset of patients with cancer(e.g., those responding to a particular therapy or those developingresistance). Diagnostic markers also include “surrogate markers,” e.g.,markers which are indirect markers of disease progression.

The term “probe” refers to any molecule which is capable of selectivelybinding to a specifically intended target molecule, for example a markerof the invention. Probes can be either synthesized by one skilled in theart, or derived from appropriate biological preparations. For purposesof detection of the target molecule, probes may be specifically designedto be labeled, as described herein. Examples of molecules that can beutilized as probes include, but are not limited to, RNA, DNA, proteins,antibodies, and organic monomers.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue-specific manner.

An “RNA interfering agent” as used herein, is defined as any agent whichinterferes with or inhibits expression of a target gene, e.g., a markerof the invention, by RNA interference (RNAi). Such RNA interferingagents include, but are not limited to, nucleic acid molecules includingRNA molecules which are homologous to the target gene, e.g., a marker ofthe invention, or a fragment thereof, short interfering RNA (siRNA), andsmall molecules which interfere with or inhibit expression of a targetgene by RNA interference (RNAi).

“RNA interference (RNAi)” is an evolutionally conserved process wherebythe expression or introduction of RNA of a sequence that is identical orhighly similar to a target gene results in the sequence specificdegradation or specific post-transcriptional gene silencing (PTGS) ofmessenger RNA (mRNA) transcribed from that targeted gene (see Coburn, G.and Cullen, B. (2002) J of Virology 76(18):9225), thereby inhibitingexpression of the target gene. In one embodiment, the RNA is doublestranded RNA (dsRNA). This process has been described in plants,invertebrates, and mammalian cells. In nature, RNAi is initiated by thedsRNA-specific endonuclease Dicer, which promotes processive cleavage oflong dsRNA into double-stranded fragments termed siRNAs. siRNAs areincorporated into a protein complex that recognizes and cleaves targetmRNAs. RNAi can also be initiated by introducing nucleic acid molecules,e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silencethe expression of target genes. As used herein, “inhibition of targetgene expression” or “inhibition of marker gene expression” includes anydecrease in expression or protein activity or level of the target gene(e.g., a marker gene of the invention) or protein encoded by the targetgene, e.g., a marker protein of the invention. The decrease may be of atleast 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as comparedto the expression of a target gene or the activity or level of theprotein encoded by a target gene which has not been targeted by an RNAinterfering agent.

“Short interfering RNA” (siRNA), also referred to herein as “smallinterfering RNA” is defined as an agent which functions to inhibitexpression of a target gene, e.g., by RNAi. An siRNA may be chemicallysynthesized, may be produced by in vitro transcription, or may beproduced within a host cell. In one embodiment, siRNA is a doublestranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides inlength, preferably about 15 to about 28 nucleotides, more preferablyabout 19 to about 25 nucleotides in length, and more preferably about19, 20, 21, or 22 nucleotides in length, and may contain a 3′ and/or 5′overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5nucleotides. The length of the overhang is independent between the twostrands, i.e., the length of the over hang on one strand is notdependent on the length of the overhang on the second strand. Preferablythe siRNA is capable of promoting RNA interference through degradationor specific post-transcriptional gene silencing (PTGS) of the targetmessenger RNA (mRNA).

In another embodiment, an siRNA is a small hairpin (also called stemloop) RNA (shRNA). In one embodiment, these shRNAs are composed of ashort (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9nucleotide loop, and the analogous sense strand. Alternatively, thesense strand may precede the nucleotide loop structure and the antisensestrand may follow. These shRNAs may be contained in plasmids,retroviruses, and lentiviruses and expressed from, for example, the polIII U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003)RNA April; 9(4):493-501 incorporated be reference herein).

RNA interfering agents, e.g., siRNA molecules, may be administered to apatient having or at risk for having cancer, to inhibit expression of amarker gene of the invention, e.g., a marker gene which is overexpressedin cancer (such as the markers listed in Table 5) and thereby treat,prevent, or inhibit cancer in the subject.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cell under mostor all physiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

A “transcribed polynucleotide” is a polynucleotide (e.g. an RNA, a cDNA,or an analog of one of an RNA or cDNA) which is complementary to orhomologous with all or a portion of a mature RNA made by transcriptionof a marker of the invention and normal post-transcriptional processing(e.g. splicing), if any, of the transcript, and reverse transcription ofthe transcript.

“Complementary” refers to the broad concept of sequence complementaritybetween regions of two nucleic acid strands or between two regions ofthe same nucleic acid strand. It is known that an adenine residue of afirst nucleic acid region is capable of forming specific hydrogen bonds(“base pairing”) with a residue of a second nucleic acid region which isantiparallel to the first region if the residue is thymine or uracil.Similarly, it is known that a cytosine residue of a first nucleic acidstrand is capable of base pairing with a residue of a second nucleicacid strand which is antiparallel to the first strand if the residue isguanine. A first region of a nucleic acid is complementary to a secondregion of the same or a different nucleic acid if, when the two regionsare arranged in an antiparallel fashion, at least one nucleotide residueof the first region is capable of base pairing with a residue of thesecond region. Preferably, the first region comprises a first portionand the second region comprises a second portion, whereby, when thefirst and second portions are arranged in an antiparallel fashion, atleast about 50%, and preferably at least about 75%, at least about 90%,or at least about 95% of the nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion. More preferably, all nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion.

The terms “homology” or “identity,” as used interchangeably herein,refer to sequence similarity between two polynucleotide sequences orbetween two polypeptide sequences, with identity being a more strictcomparison. The phrases “percent identity or homology” and “% identityor homology” refer to the percentage of sequence similarity found in acomparison of two or more polynucleotide sequences or two or morepolypeptide sequences. “Sequence similarity” refers to the percentsimilarity in base pair sequence (as determined by any suitable method)between two or more polynucleotide sequences. Two or more sequences canbe anywhere from 0-100% similar, or any integer value there between.Identity or similarity can be determined by comparing a position in eachsequence that may be aligned for purposes of comparison. When a positionin the compared sequence is occupied by the same nucleotide base oramino acid, then the molecules are identical at that position. A degreeof similarity or identity between polynucleotide sequences is a functionof the number of identical or matching nucleotides at positions sharedby the polynucleotide sequences. A degree of identity of polypeptidesequences is a function of the number of identical amino acids atpositions shared by the polypeptide sequences. A degree of homology orsimilarity of polypeptide sequences is a function of the number of aminoacids at positions shared by the polypeptide sequences. The term“substantial homology,” as used herein, refers to homology of at least50%, more preferably, 60%, 70%, 80%, 90%, 95% or more.

A marker is “fixed” to a substrate if it is covalently or non-covalentlyassociated with the substrate such the substrate can be rinsed with afluid (e.g. standard saline citrate, pH 7.4) without a substantialfraction of the marker dissociating from the substrate.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g. encodes a natural protein).

Cancer is “inhibited” if at least one symptom of the cancer isalleviated, terminated, slowed, or prevented. As used herein, cancer isalso “inhibited” if recurrence or metastasis of the cancer is reduced,slowed, delayed, or prevented.

A kit is any manufacture (e.g. a package or container) comprising atleast one reagent, e.g. a probe, for specifically detecting a marker ofthe invention, the manufacture being promoted, distributed, or sold as aunit for performing the methods of the present invention.

II. Uses of the Invention

The present invention is based, in part, on the identification ofchromosomal regions (MCRs) which are structurally altered leading to adifferent copy number in cancer cells as compared to normal (i.e.non-cancerous) cells. Furthermore, the present invention is based, inpart, on the identification of markers, e.g., markers which reside inthe MCRs of the invention, which have an altered amount, structure,and/or activity in cancer cells as compared to normal (i.e.,non-cancerous) cells. The markers of the invention correspond to DNA,cDNA, RNA, and polypeptide molecules which can be detected in one orboth of normal and cancerous cells.

The amount, structure, and/or activity, e.g., the presence, absence,copy number, expression level, protein level, protein activity, presenceof mutations, e.g., mutations which affect activity of the marker (e.g.,substitution, deletion, or addition mutations), and/or methylationstatus, of one or more of these markers in a sample, e.g., a samplecontaining tissue, whole blood, serum, plasma, buccal scrape, saliva,cerebrospinal fluid, urine, stool, bile, pancreatic juice, andpancreatic tissue, is herein correlated with the cancerous state of thetissue. In addition, the presence, absence, and/or copy number of one ormore of the MCRs of the invention in a sample is also correlated withthe cancerous state of the tissue. The invention thus providescompositions, kits, and methods for assessing the cancerous state ofcells (e.g. cells obtained from a non-human, cultured non-human cells,and in vivo cells) as well as methods for treatment, prevention, and/orinhibition of cancer using a modulator, e.g., an agonist or antagonist,of a marker of the invention.

The compositions, kits, and methods of the invention have the followinguses, among others:

-   -   1) assessing whether a subject is afflicted with cancer;    -   2) assessing the stage of cancer in a human subject;    -   3) assessing the grade of cancer in a subject;    -   4) assessing the benign or malignant nature of cancer in a        subject;    -   5) assessing the metastatic potential of cancer in a subject;    -   6) assessing the histological type of neoplasm associated with        cancer in a subject;    -   7) making antibodies, antibody fragments or antibody derivatives        that are useful for treating cancer and/or assessing whether a        subject is afflicted with cancer;    -   8) assessing the presence of cancer cells;    -   9) assessing the efficacy of one or more test compounds for        inhibiting cancer in a subject;    -   10) assessing the efficacy of a therapy for inhibiting cancer in        a subject;    -   11) monitoring the progression of cancer in a subject;    -   12) selecting a composition or therapy for inhibiting cancer,        e.g., in a subject;    -   13) treating a subject afflicted with cancer;    -   14) inhibiting cancer in a subject;    -   15) assessing the carcinogenic potential of a test compound; and    -   16) preventing the onset of cancer in a subject at risk for        developing cancer.

The invention thus includes a method of assessing whether a subject isafflicted with cancer or is at risk for developing cancer. This methodcomprises comparing the amount, structure, and/or activity, e.g., thepresence, absence, copy number, expression level, protein level, proteinactivity, presence of mutations, e.g., mutations which affect activityof the marker (e.g., substitution, deletion, or addition mutations),and/or methylation status, of a marker in a subject sample with thenormal level. A significant difference between the amount, structure, oractivity of the marker in the subject sample and the normal level is anindication that the subject is afflicted with cancer. The invention alsoprovides a method for assessing whether a subject is afflicted withcancer or is at risk for developing cancer by comparing the level ofexpression of marker(s) within an MCR or copy number of an MCR in acancer sample with the level of expression of marker(s) within an MCR orcopy number of an MCR in a normal, control sample. A significantdifference between the level of expression of marker(s) within an MCR orcopy number of the MCR in the subject sample and the normal level is anindication that the subject is afflicted with cancer. The MCR isselected from the group consisting of those listed in Table 1.

The marker is selected from the group consisting of the markers listedin Tables 4 and 5. Table 4 lists markers which have a highly significantcorrelation between gene expression and gene dosage (p, 0.05). The levelof expression or copy number of these markers is decreased in sampleshistologically identified as pancreatic cancer, e.g., pancreaticadenocarcinoma. Table 4 also lists the chromosome, physical position inMb, Gene Weight, p-value, Affymetrix™ probe(s) number corresponding toeach UniGene ID, Genebank Accession No. (i.e., “GI” number), and SEQ IDNO. for each of the markers. Although one or more moleculescorresponding to the markers listed in Table 4 may have been describedby others, the significance of these markers with regard to thecancerous state of cells, has not previously been identified.

Table 5 also lists markers which have a highly significant correlationbetween gene expression and gene dosage (p, 0.05). The level ofexpression or copy number of these markers is increased in sampleshistologically identified as pancreatic cancer, e.g., pancreaticadenocarcinoma. Table 5 also lists the chromosome, physical position inMb, Gene Weight, p-value, Affymetrix™ probe(s) number corresponding toeach UniGene ID, Genebank Accession No. (i.e., “GI” number), and SEQ IDNO. for each of these markers. Although one or more moleculescorresponding to the markers listed in Table 5 may have been describedby others, the significance of these markers with regard to thecancerous state of cells, has not previously been identified.

Any marker or combination of markers listed in Tables 4 or 5 or any MCRor combination of MCRs listed in Table 1, may be used in thecompositions, kits, and methods of the present invention. In general, itis preferable to use markers for which the difference between theamount, e.g., level of expression or copy number, and/or activity of themarker or MCR in cancer cells and the amount, e.g., level of expressionor copy number, and/or activity of the same marker in normal cells, isas great as possible. Although this difference can be as small as thelimit of detection of the method for assessing amount and/or activity ofthe marker, it is preferred that the difference be at least greater thanthe standard error of the assessment method, and preferably a differenceof at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 100-,500-, 1000-fold or greater than the amount, e.g., level of expression orcopy number, and/or activity of the same biomarker in normal tissue.

It is understood that by routine screening of additional subject samplesusing one or more of the markers of the invention, it will be realizedthat certain of the markers have altered amount, structure, and/oractivity in cancers of various types, including specific pancreaticcancers, as well as other cancers, e.g., carcinoma, sarcoma, lymphoma orleukemia, examples of which include, but are not limited to, melanomas,breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostatecancer, pancreas cancer, stomach cancer, ovarian cancer, urinary bladdercancer, brain or central nervous system cancer, peripheral nervoussystem cancer, esophageal cancer, cervical cancer, uterine orendometrial cancer, cancer of the oral cavity or pharynx, liver cancer,kidney cancer, testicular cancer, biliary tract cancer, small bowel orappendix cancer, salivary gland cancer, thyroid gland cancer, adrenalgland cancer, osteosarcoma, chondrosarcoma, cancer of hematologicaltissues, and the like.

For example, it will be confirmed that some of the markers of theinvention have altered amount, structure, and/or activity in some, i.e.,10%, 20%, 30%, or 40%, or most (i.e. 50% or more) or substantially all(i.e. 80% or more) of cancer, e.g., pancreatic cancer. Furthermore, itwill be confirmed that certain of the markers of the invention areassociated with cancer of various histologic subtypes.

In addition, as a greater number of subject samples are assessed foraltered amount, structure, and/or activity of the markers or alteredexpression or copy number MCRs of the invention and the outcomes of theindividual subjects from whom the samples were obtained are correlated,it will also be confirmed that markers have altered amount, structure,and/or activity of certain of the markers or altered expression or copynumber of MCRs of the invention are strongly correlated with malignantcancers and that altered expression of other markers of the inventionare strongly correlated with benign tumors or premalignant states. Thecompositions, kits, and methods of the invention are thus useful forcharacterizing one or more of the stage, grade, histological type, andbenign/premalignant/malignant nature of cancer in subjects.

When the compositions, kits, and methods of the invention are used forcharacterizing one or more of the stage, grade, histological type, andbenign/premalignant/malignant nature of cancer, in a subject, it ispreferred that the marker or MCR or panel of markers or MCRs of theinvention be selected such that a positive result is obtained in atleast about 20%, and preferably at least about 40%, 60%, or 80%, andmore preferably, in substantially all, subjects afflicted with cancer,of the corresponding stage, grade, histological type, orbenign/premalignant/malignant nature. Preferably, the marker or panel ofmarkers of the invention is selected such that a PPV (positivepredictive value) of greater than about 10% is obtained for the generalpopulation (more preferably coupled with an assay specificity greaterthan 99.5%).

When a plurality of markers or MCRs of the invention are used in thecompositions, kits, and methods of the invention, the amount, structure,and/or activity of each marker or level of expression or copy number canbe compared with the normal amount, structure, and/or activity of eachof the plurality of markers or level of expression or copy number, innon-cancerous samples of the same type, either in a single reactionmixture (i.e. using reagents, such as different fluorescent probes, foreach marker) or in individual reaction mixtures corresponding to one ormore of the markers or MCRs.

In one embodiment, a significantly altered amount, structure, and/oractivity of more than one of the plurality of markers, or significantlyaltered copy number of one or more of the MCRs in the sample, relativeto the corresponding normal levels, is an indication that the subject isafflicted with cancer. For example, a significantly lower copy number inthe sample of each of the plurality of markers or MCRs, relative to thecorresponding normal levels or copy number, is an indication that thesubject is afflicted with cancer. In yet another embodiment, asignificantly enhanced copy number of one or more markers or MCRs and asignificantly lower level of expression or copy number of one or moremarkers or MCRs in a sample relative to the corresponding normal levels,is an indication that the subject is afflicted with cancer. Also, forexample, a significantly enhanced copy number in the sample of each ofthe plurality of markers or MCRs, relative to the corresponding normalcopy number, is an indication that the subject is afflicted with cancer.In yet another embodiment, a significantly enhanced copy number of oneor more markers or MCRs and a significantly lower copy number of one ormore markers or MCRs in a sample relative to the corresponding normallevels, is an indication that the subject is afflicted with cancer.

When a plurality of markers or MCRs are used, it is preferred that 2, 3,4, 5, 8, 10, 12, 15, 20, 30, or 50 or more individual markers or MCRs beused or identified, wherein fewer markers or MCRs are preferred.

Only a small number of markers are known to be associated with, forexample, pancreatic cancer (e.g., AKT2, p16^(INK4a), c-MYC, SMAD4, andTP53; Lynch, supra). These markers or other markers which are known tobe associated with other types of cancer may be used together with oneor more markers of the invention in, for example, a panel of markers. Inaddition, frequent gains have been mapped to 3q, 5p, 7p, 8q, 11q, 12p,17q and 20q and losses to 3p, 4q, 6q, 8p, 9p, 10q, 12q, 13q, 17p, 18qand 21q and 22q in pancreatic cancer. In some instances, validatedoncogenes and tumor suppressor genes residing within these loci havebeen identified, including MYC (8q24), p16^(INK4A) (9p21), p53 (17p13),SMAD4 (18q21) and AKT2 (19q13). It is well known that certain types ofgenes, such as oncogenes, tumor suppressor genes, growth factor-likegenes, protease-like genes, and protein kinase-like genes are ofteninvolved with development of cancers of various types. Thus, among themarkers of the invention, use of those which correspond to proteinswhich resemble known proteins encoded by known oncogenes and tumorsuppressor genes, and those which correspond to proteins which resemblegrowth factors, proteases, and protein kinases, are preferred.

It is recognized that the compositions, kits, and methods of theinvention will be of particular utility to subjects having an enhancedrisk of developing cancer, and their medical advisors. Subjectsrecognized as having an enhanced risk of developing cancer, include, forexample, subjects having a familial history of cancer, subjectsidentified as having a mutant oncogene (i.e. at least one allele), andsubjects of advancing age.

An alteration, e.g. copy number, amount, structure, and/or activity of amarker in normal (i.e. non-cancerous) human tissue can be assessed in avariety of ways. In one embodiment, the normal level of expression orcopy number is assessed by assessing the level of expression and/or copynumber of the marker or MCR in a portion of cells which appear to benon-cancerous and by comparing this normal level of expression or copynumber with the level of expression or copy number in a portion of thecells which are suspected of being cancerous. For example, whenlaparoscopy or other medical procedure, reveals the presence of a tumoron one portion of an organ, the normal level of expression or copynumber of a marker or MCR may be assessed using the non-affected portionof the organ, and this normal level of expression or copy number may becompared with the level of expression or copy number of the same markerin an affected portion (i.e., the tumor) of the organ. Alternately, andparticularly as further information becomes available as a result ofroutine performance of the methods described herein, population-averagevalues for “normal” copy number, amount, structure, and/or activity ofthe markers or MCRs of the invention may be used. In other embodiments,the “normal” copy number, amount, structure, and/or activity of a markeror MCR may be determined by assessing copy number, amount, structure,and/or activity of the marker or MCR in a subject sample obtained from anon-cancer-afflicted subject, from a subject sample obtained from asubject before the suspected onset of cancer in the subject, fromarchived subject samples, and the like.

The invention includes compositions, kits, and methods for assessing thepresence of cancer cells in a sample (e.g. an archived tissue sample ora sample obtained from a subject). These compositions, kits, and methodsare substantially the same as those described above, except that, wherenecessary, the compositions, kits, and methods are adapted for use withcertain types of samples. For example, when the sample is a parafinized,archived human tissue sample, it may be necessary to adjust the ratio ofcompounds in the compositions of the invention, in the kits of theinvention, or the methods used. Such methods are well known in the artand within the skill of the ordinary artisan.

The invention thus includes a kit for assessing the presence of cancercells (e.g. in a sample such as a subject sample). The kit may compriseone or more reagents capable of identifying a marker or MCR of theinvention, e.g., binding specifically with a nucleic acid or polypeptidecorresponding to a marker or MCR of the invention. Suitable reagents forbinding with a polypeptide corresponding to a marker of the inventioninclude antibodies, antibody derivatives, antibody fragments, and thelike. Suitable reagents for binding with a nucleic acid (e.g. a genomicDNA, an mRNA, a spliced mRNA, a cDNA, or the like) include complementarynucleic acids. For example, the nucleic acid reagents may includeoligonucleotides (labeled or non-labeled) fixed to a substrate, labeledoligonucleotides not bound with a substrate, pairs of PCR primers,molecular beacon probes, and the like.

The kit of the invention may optionally comprise additional componentsuseful for performing the methods of the invention. By way of example,the kit may comprise fluids (e.g., SSC buffer) suitable for annealingcomplementary nucleic acids or for binding an antibody with a proteinwith which it specifically binds, one or more sample compartments, aninstructional material which describes performance of a method of theinvention, a sample of normal cells, a sample of cancer cells, and thelike.

A kit of the invention may comprise a reagent useful for determiningprotein level or protein activity of a marker. In another embodiment, akit of the invention may comprise a reagent for determining methylationstatus of a marker, or may comprise a reagent for determining alterationof structure of a marker, e.g., the presence of a mutation.

The invention also includes a method of making an isolated hybridomawhich produces an antibody useful in methods and kits of the presentinvention. A protein corresponding to a marker of the invention may beisolated (e.g. by purification from a cell in which it is expressed orby transcription and translation of a nucleic acid encoding the proteinin vivo or in vitro using known methods) and a vertebrate, preferably amammal such as a mouse, rat, rabbit, or sheep, is immunized using theisolated protein. The vertebrate may optionally (and preferably) beimmunized at least one additional time with the isolated protein, sothat the vertebrate exhibits a robust immune response to the protein.Splenocytes are isolated from the immunized vertebrate and fused with animmortalized cell line to form hybridomas, using any of a variety ofmethods well known in the art. Hybridomas formed in this manner are thenscreened using standard methods to identify one or more hybridomas whichproduce an antibody which specifically binds with the protein. Theinvention also includes hybridomas made by this method and antibodiesmade using such hybridomas.

The invention also includes a method of assessing the efficacy of a testcompound for inhibiting cancer cells. As described above, differences inthe amount, structure, and/or activity of the markers of the invention,or level of expression or copy number of the MCRs of the invention,correlate with the cancerous state of cells. Although it is recognizedthat changes in the levels of amount, e.g., expression or copy number,structure, and/or activity of certain of the markers or expression orcopy number of the MCRs of the invention likely result from thecancerous state of cells, it is likewise recognized that changes in theamount may induce, maintain, and promote the cancerous state. Thus,compounds which inhibit cancer, in a subject may cause a change, e.g., achange in expression and/or activity of one or more of the markers ofthe invention to a level nearer the normal level for that marker (e.g.,the amount, e.g., expression, and/or activity for the marker innon-cancerous cells).

This method thus comprises comparing amount, e.g., expression, and/oractivity of a marker in a first cell sample and maintained in thepresence of the test compound and amount, e.g., expression, and/oractivity of the marker in a second cell sample and maintained in theabsence of the test compound. A significant increase in the amount,e.g., expression, and/or activity of a marker listed in Table 4 (e.g., amarker that was shown to be decreased in cancer), a significant decreasein the amount, e.g., expression, and/or activity of a marker listed inTable 5 (e.g., a marker that was shown to be increased in cancer), is anindication that the test compound inhibits cancer. The cell samples may,for example, be aliquots of a single sample of normal cells obtainedfrom a subject, pooled samples of normal cells obtained from a subject,cells of a normal cell lines, aliquots of a single sample of cancer,cells obtained from a subject, pooled samples of cancer, cells obtainedfrom a subject, cells of a cancer cell line, cells from an animal modelof cancer, or the like. In one embodiment, the samples are cancer cellsobtained from a subject and a plurality of compounds known to beeffective for inhibiting various cancers, are tested in order toidentify the compound which is likely to best inhibit the cancer in thesubject.

This method may likewise be used to assess the efficacy of a therapy,e.g., chermotherapy, radiation therapy, surgery, or any othertherapeutic approach useful for inhibiting cancer in a subject. In thismethod, the amount, e.g., expression, and/or activity of one or moremarkers of the invention in a pair of samples (one subjected to thetherapy, the other not subjected to the therapy) is assessed. As withthe method of assessing the efficacy of test compounds, if the therapyinduces a significant decrease in the amount, e.g., expression, and/oractivity of a marker listed in Table 5 (e.g., a marker that was shown tobe increased in cancer), blocks induction of a marker listed in Table 5(e.g., a marker that was shown to be increased in cancer), or if thetherapy induces a significant enhancement of the amount, e.g.,expression, and/or activity of a marker listed in Table 4 (e.g., amarker that was shown to be decreased in cancer), then the therapy isefficacious for inhibiting cancer. As above, if samples from a selectedsubject are used in this method, then alternative therapies can beassessed in vitro in order to select a therapy most likely to beefficacious for inhibiting cancer in the subject.

This method may likewise be used to monitor the progression of cancer ina subject, wherein if a sample in a subject has a significant decreasein the amount, e.g., expression, and/or activity of a marker listed inTable 5 (e.g., a marker that was shown to be increased in cancer, orblocks induction of a marker listed in Table 5 (e.g., a marker that wasshown to be increased in cancer), or a significant enhancement of theamount, e.g., expression, and/or activity of a marker listed in Table 4(e.g., a marker that was shown to be decreased in cancer), during theprogression of cancer, e.g., at a first point in time and a subsequentpoint in time, then the cancer has improved. In yet another embodiment,between the first point in time and a subsequent point in time, thesubject has undergone treatment, e.g., chermotherapy, radiation therapy,surgery, or any other therapeutic approach useful for inhibiting cancer,has completed treatment, or is in remission.

As described herein, cancer in subjects is associated with an increasein amount, e.g., expression, and/or activity of one or more markerslisted in Table 5 (e.g., a marker that was shown to be increased incancer), and/or a decrease in amount, e.g., expression, and/or activityof one or more markers listed in Table 4 (e.g., a marker that was shownto be decreased in cancer). While, as discussed above, some of thesechanges in amount, e.g., expression, and/or activity number result fromoccurrence of the cancer, others of these changes induce, maintain, andpromote the cancerous state of cancer cells. Thus, cancer characterizedby an increase in the amount, e.g., expression, and/or activity of oneor more markers listed in Table 5 (e.g., a marker that was shown to beincreased in cancer), can be inhibited by inhibiting amount, e.g.,expression, and/or activity of those markers. Likewise, cancercharacterized by a decrease in the amount, e.g., expression, and/oractivity of one or more markers listed in Table 4 (e.g., a marker thatwas shown to be decreased in cancer), can be inhibited by enhancingamount, e.g., expression, and/or activity of those markers.

Amount and/or activity of a marker listed in Table 5 (e.g., a markerthat was shown to be increased in cancer), can be inhibited in a numberof ways generally known in the art. For example, an antisenseoligonucleotide can be provided to the cancer cells in order to inhibittranscription, translation, or both, of the marker(s). An RNAinterfering agent, e.g., an siRNA molecule, which is targeted to amarker listed in Table 5, can be provided to the cancer cells in orderto inhibit expression of the target marker, e.g., through degradation orspecific post-transcriptional gene silencing (PTGS) of the messenger RNA(mRNA) of the target marker. Alternately, a polynucleotide encoding anantibody, an antibody derivative, or an antibody fragment, e.g., afragment capable of binding an antigen, and operably linked with anappropriate promoter or regulator region, can be provided to the cell inorder to generate intracellular antibodies which will inhibit thefunction, amount, and/or activity of the protein corresponding to themarker(s). Conjugated antibodies or fragments thereof, e.g.,chemolabeled antibodies, radiolabeled antibodies, or immunotoxinstargeting a marker of the invention may also be administered to treat,prevent or inhibit cancer.

A small molecule may also be used to modulate, e.g., inhibit, expressionand/or activity of a marker listed in Table 5. In one embodiment, asmall molecule functions to disrupt a protein-protein interactionbetween a marker of the invention and a target molecule or ligand,thereby modulating, e.g., increasing or decreasing the activity of themarker.

Using the methods described herein, a variety of molecules, particularlyincluding molecules sufficiently small that they are able to cross thecell membrane, can be screened in order to identify molecules whichinhibit amount and/or activity of the marker(s). The compound soidentified can be provided to the subject in order to inhibit amountand/or activity of the marker(s) in the cancer cells of the subject.

Amount and/or activity of a marker listed in Table 4 (e.g., a markerthat was shown to be decreased in cancer), can be enhanced in a numberof ways generally known in the art. For example, a polynucleotideencoding the marker and operably linked with an appropriatepromoter/regulator region can be provided to cells of the subject inorder to induce enhanced expression and/or activity of the protein (andmRNA) corresponding to the marker therein. Alternatively, if the proteinis capable of crossing the cell membrane, inserting itself in the cellmembrane, or is normally a secreted protein, then amount and/or activityof the protein can be enhanced by providing the protein (e.g. directlyor by way of the bloodstream) to cancer cells in the subject. A smallmolecule may also be used to modulate, e.g., increase, expression oractivity of a marker listed in Table 4. Furthermore, in anotherembodiment, a modulator of a marker of the invention, e.g., a smallmolecule, may be used, for example, to re-express a silenced gene, e.g.,a tumor suppressor, in order to treat or prevent cancer. For example,such a modulator may interfere with a DNA binding element or amethyltransferase.

As described above, the cancerous state of human cells is correlatedwith changes in the amount and/or activity of the markers of theinvention. Thus, compounds which induce increased expression or activityof one or more of the markers listed in Table 5 (e.g., a marker that wasshown to be increased in cancer), decreased amount and/or activity ofone or more of the markers listed in Table 4 (e.g., a marker that wasshown to be decreased in cancer), can induce cell carcinogenesis. Theinvention also includes a method for assessing the human cellcarcinogenic potential of a test compound. This method comprisesmaintaining separate aliquots of human cells in the presence and absenceof the test compound. Expression or activity of a marker of theinvention in each of the aliquots is compared. A significant increase inthe amount and/or activity of a marker listed in Table 5 (e.g., a markerthat was shown to be increased in cancer), or a significant decrease inthe amount and/or activity of a marker listed in Table 4 (e.g., a markerthat was shown to be decreased in cancer), in the aliquot maintained inthe presence of the test compound (relative to the aliquot maintained inthe absence of the test compound) is an indication that the testcompound possesses human cell carcinogenic potential. The relativecarcinogenic potentials of various test compounds can be assessed bycomparing the degree of enhancement or inhibition of the amount and/oractivity of the relevant markers, by comparing the number of markers forwhich the amount and/or activity is enhanced or inhibited, or bycomparing both.

Various aspects of the invention are described in further detail in thefollowing subsections.

III. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat correspond to a marker of the invention, including nucleic acidswhich encode a polypeptide corresponding to a marker of the invention ora portion of such a polypeptide. The nucleic acid molecules of theinvention include those nucleic acid molecules which reside in the MCRsidentified herein. Isolated nucleic acid molecules of the invention alsoinclude nucleic acid molecules sufficient for use as hybridizationprobes to identify nucleic acid molecules that correspond to a marker ofthe invention, including nucleic acid molecules which encode apolypeptide corresponding to a marker of the invention, and fragments ofsuch nucleic acid molecules, e.g., those suitable for use as PCR primersfor the amplification or mutation of nucleic acid molecules. As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. Preferably, an “isolated” nucleic acid moleculeis free of sequences (preferably protein-encoding sequences) whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated nucleic acid molecule can contain less than about 5 kB, 4kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecules encoding a protein corresponding to a marker listed in Tables4 or 5, can be isolated using standard molecular biology techniques andthe sequence information in the database records described herein. Usingall or a portion of such nucleic acid sequences, nucleic acid moleculesof the invention can be isolated using standard hybridization andcloning techniques (e.g., as described in Sambrook et al., ed.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA, or genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid molecules so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to all or a portion of a nucleic acid molecule of theinvention can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which has a nucleotidesequence complementary to the nucleotide sequence of a nucleic acidcorresponding to a marker of the invention or to the nucleotide sequenceof a nucleic acid encoding a protein which corresponds to a marker ofthe invention. A nucleic acid molecule which is complementary to a givennucleotide sequence is one which is sufficiently complementary to thegiven nucleotide sequence that it can hybridize to the given nucleotidesequence thereby forming a stable duplex.

Moreover, a nucleic acid molecule of the invention can comprise only aportion of a nucleic acid sequence, wherein the full length nucleic acidsequence comprises a marker of the invention or which encodes apolypeptide corresponding to a marker of the invention. Such nucleicacid molecules can be used, for example, as a probe or primer. Theprobe/primer typically is used as one or more substantially purifiedoligonucleotides. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 7, preferably about 15, more preferably about 25, 50, 75,100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutivenucleotides of a nucleic acid of the invention.

Probes based on the sequence of a nucleic acid molecule of the inventioncan be used to detect transcripts or genomic sequences corresponding toone or more markers of the invention. The probe comprises a label groupattached thereto, e.g., a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used as part of adiagnostic test kit for identifying cells or tissues which mis-expressthe protein, such as by measuring levels of a nucleic acid moleculeencoding the protein in a sample of cells from a subject, e.g.,detecting mRNA levels or determining whether a gene encoding the proteinhas been mutated or deleted.

The invention further encompasses nucleic acid molecules that differ,due to degeneracy of the genetic code, from the nucleotide sequence ofnucleic acid molecules encoding a protein which corresponds to a markerof the invention, and thus encode the same protein.

In addition to the nucleotide sequences described in Tables 4 or 5, itwill be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequence can existwithin a population (e.g., the human population). Such geneticpolymorphisms can exist among individuals within a population due tonatural allelic variation. An allele is one of a group of genes whichoccur alternatively at a given genetic locus. In addition, it will beappreciated that DNA polymorphisms that affect RNA expression levels canalso exist that may affect the overall expression level of that gene(e.g., by affecting regulation or degradation).

As used herein, the phrase “allelic variant” refers to a nucleotidesequence which occurs at a given locus or to a polypeptide encoded bythe nucleotide sequence.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a polypeptidecorresponding to a marker of the invention. Such natural allelicvariations can typically result in 1-5% variance in the nucleotidesequence of a given gene. Alternative alleles can be identified bysequencing the gene of interest in a number of different individuals.This can be readily carried out by using hybridization probes toidentify the same genetic locus in a variety of individuals. Any and allsuch nucleotide variations and resulting amino acid polymorphisms orvariations that are the result of natural allelic variation and that donot alter the functional activity are intended to be within the scope ofthe invention.

In another embodiment, an isolated nucleic acid molecule of theinvention is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250,300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600,1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, or morenucleotides in length and hybridizes under stringent conditions to anucleic acid molecule corresponding to a marker of the invention or to anucleic acid molecule encoding a protein corresponding to a marker ofthe invention. As used herein, the term “hybridizes under stringentconditions” is intended to describe conditions for hybridization andwashing under which nucleotide sequences at least 60% (65%, 70%,preferably 75%) identical to each other typically remain hybridized toeach other. Such stringent conditions are known to those skilled in theart and can be found in sections 6.3.1-6.3.6 of Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989). A preferred,non-limiting example of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.

In addition to naturally-occurring allelic variants of a nucleic acidmolecule of the invention that can exist in the population, the skilledartisan will further appreciate that sequence changes can be introducedby mutation thereby leading to changes in the amino acid sequence of theencoded protein, without altering the biological activity of the proteinencoded thereby. For example, one can make nucleotide substitutionsleading to amino acid substitutions at “non-essential” amino acidresidues. A “non-essential” amino acid residue is a residue that can bealtered from the wild-type sequence without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. For example, amino acid residues that are notconserved or only semi-conserved among homologs of various species maybe non-essential for activity and thus would be likely targets foralteration. Alternatively, amino acid residues that are conserved amongthe homologs of various species (e.g., murine and human) may beessential for activity and thus would not be likely targets foralteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding a polypeptide of the invention that contain changesin amino acid residues that are not essential for activity. Suchpolypeptides differ in amino acid sequence from the naturally-occurringproteins which correspond to the markers of the invention, yet retainbiological activity. In one embodiment, such a protein has an amino acidsequence that is at least about 40% identical, 50%, 60%, 70%, 80%, 90%,95%, or 98% identical to the amino acid sequence of one of the proteinswhich correspond to the markers of the invention.

An isolated nucleic acid molecule encoding a variant protein can becreated by introducing one or more nucleotide substitutions, additionsor deletions into the nucleotide sequence of nucleic acids of theinvention, such that one or more amino acid residue substitutions,additions, or deletions are introduced into the encoded protein.Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined.

The present invention encompasses antisense nucleic acid molecules,i.e., molecules which are complementary to a sense nucleic acid of theinvention, e.g., complementary to the coding strand of a double-strandedcDNA molecule corresponding to a marker of the invention orcomplementary to an mRNA sequence corresponding to a marker of theinvention. Accordingly, an antisense nucleic acid molecule of theinvention can hydrogen bond to (i.e. anneal with) a sense nucleic acidof the invention. The antisense nucleic acid can be complementary to anentire coding strand, or to only a portion thereof, e.g., all or part ofthe protein coding region (or open reading frame). An antisense nucleicacid molecule can also be antisense to all or part of a non-codingregion of the coding strand of a nucleotide sequence encoding apolypeptide of the invention. The non-coding regions (“5′ and 3′untranslated regions”) are the 5′ and 3′ sequences which flank thecoding region and are not translated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45, or 50 or more nucleotides in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been sub-cloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a polypeptidecorresponding to a selected marker of the invention to thereby inhibitexpression of the marker, e.g., by inhibiting transcription and/ortranslation. The hybridization can be by conventional nucleotidecomplementarity to form a stable duplex, or, for example, in the case ofan antisense nucleic acid molecule which binds to DNA duplexes, throughspecific interactions in the major groove of the double helix. Examplesof a route of administration of antisense nucleic acid molecules of theinvention includes direct injection at a tissue site or infusion of theantisense nucleic acid into an ovary-associated body fluid.Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense molecules can be modified suchthat they specifically bind to receptors or antigens expressed on aselected cell surface, e.g., by linking the antisense nucleic acidmolecules to peptides or antibodies which bind to cell surface receptorsor antigens. The antisense nucleic acid molecules can also be deliveredto cells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual α-units, the strands run parallel to each other(Gaultier et al., 1987, Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

The invention also encompasses ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes asdescribed in Haselhoff and Gerlach, 1988, Nature 334:585-591) can beused to catalytically cleave mRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. A ribozyme havingspecificity for a nucleic acid molecule encoding a polypeptidecorresponding to a marker of the invention can be designed based uponthe nucleotide sequence of a cDNA corresponding to the marker. Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved (see Cech et al. U.S. Pat. No.4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). Alternatively, anmRNA encoding a polypeptide of the invention can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules (see, e.g., Bartel and Szostak, 1993, Science 261:1411-1418).

The invention also encompasses nucleic acid molecules which form triplehelical structures. For example, expression of a polypeptide of theinvention can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the gene encoding thepolypeptide (e.g., the promoter and/or enhancer) to form triple helicalstructures that prevent transcription of the gene in target cells. Seegenerally Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992)Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays14(12):807-15.

In various embodiments, the nucleic acid molecules of the invention canbe modified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacid molecules can be modified to generate peptide nucleic acidmolecules (see Hyrup et al., 1996, Bioorganic & Medicinal Chemistry4(1): 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs”refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribosephosphate backbone is replaced by a pseudopeptide backbone and only thefour natural nucleobases are retained. The neutral backbone of PNAs hasbeen shown to allow for specific hybridization to DNA and RNA underconditions of low ionic strength. The synthesis of PNA oligomers can beperformed using standard solid phase peptide synthesis protocols asdescribed in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996)Proc. Natl. Acad. Sci. USA 93:14670-675.

PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs canalso be used, e.g., in the analysis of single base pair mutations in agene by, e.g., PNA directed PCR clamping; as artificial restrictionenzymes when used in combination with other enzymes, e.g., S1 nucleases(Hyrup (1996), supra; or as probes or primers for DNA sequence andhybridization (Hyrup, 1996, supra; Perry-O'Keefe et al., 1996, Proc.Natl. Acad. Sci. USA 93:14670-675).

In another embodiment, PNAs can be modified, e.g., to enhance theirstability or cellular uptake, by attaching lipophilic or other helpergroups to PNA, by the formation of PNA-DNA chimeras, or by the use ofliposomes or other techniques of drug delivery known in the art. Forexample, PNA-DNA chimeras can be generated which can combine theadvantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNASE H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup, 1996, supra). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.For example, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry and modified nucleosideanalogs. Compounds such as 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite can be used as a link between the PNA and the 5′ end ofDNA (Mag et al., 1989, Nucleic Acids Res. 17:5973-88). PNA monomers arethen coupled in a step-wise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment (Finn et al., 1996, Nucleic AcidsRes. 24(17):3357-63). Alternatively, chimeric molecules can besynthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al.,1975, Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide can include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al., 1988, Bio/Techniques 6:958-976) or intercalating agents (see,e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide can be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The invention also includes molecular beacon nucleic acid moleculeshaving at least one region which is complementary to a nucleic acidmolecule of the invention, such that the molecular beacon is useful forquantitating the presence of the nucleic acid molecule of the inventionin a sample. A “molecular beacon” nucleic acid is a nucleic acidmolecule comprising a pair of complementary regions and having afluorophore and a fluorescent quencher associated therewith. Thefluorophore and quencher are associated with different portions of thenucleic acid in such an orientation that when the complementary regionsare annealed with one another, fluorescence of the fluorophore isquenched by the quencher. When the complementary regions of the nucleicacid molecules are not annealed with one another, fluorescence of thefluorophore is quenched to a lesser degree. Molecular beacon nucleicacid molecules are described, for example, in U.S. Pat. No. 5,876,930.

IV. Isolated Proteins and Antibodies

One aspect of the invention pertains to isolated proteins whichcorrespond to individual markers of the invention, and biologicallyactive portions thereof, as well as polypeptide fragments suitable foruse as immunogens to raise antibodies directed against a polypeptidecorresponding to a marker of the invention. In one embodiment, thenative polypeptide corresponding to a marker can be isolated from cellsor tissue sources by an appropriate purification scheme using standardprotein purification techniques. In another embodiment, polypeptidescorresponding to a marker of the invention are produced by recombinantDNA techniques. Alternative to recombinant expression, a polypeptidecorresponding to a marker of the invention can be synthesized chemicallyusing standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”). When the protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When the protein isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the proteinhave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest.

Biologically active portions of a polypeptide corresponding to a markerof the invention include polypeptides comprising amino acid sequencessufficiently identical to or derived from the amino acid sequence of theprotein corresponding to the marker (e.g., the protein encoded by thenucleic acid molecules listed in Tables 4 or 5), which include feweramino acids than the full length protein, and exhibit at least oneactivity of the corresponding full-length protein. Typically,biologically active portions comprise a domain or motif with at leastone activity of the corresponding protein. A biologically active portionof a protein of the invention can be a polypeptide which is, forexample, 10, 25, 50, 100 or more amino acids in length. Moreover, otherbiologically active portions, in which other regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated for oneor more of the functional activities of the native form of a polypeptideof the invention.

Preferred polypeptides have an amino acid sequence of a protein encodedby a nucleic acid molecule listed in Tables 4 or 5. Other usefulproteins are substantially identical (e.g., at least about 40%,preferably 50%, 60%, 70%, 80%, 90%, 95%, or 99%) to one of thesesequences and retain the functional activity of the protein of thecorresponding naturally-occurring protein yet differ in amino acidsequence due to natural allelic variation or mutagenesis.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity =#ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, (1988) Comput Appl Biosci, 4:11-7. Suchan algorithm is incorporated into the ALIGN program (version 2.0) whichis part of the GCG sequence alignment software package. When utilizingthe ALIGN program for comparing amino acid sequences, a PAM120 weightresidue table, a gap length penalty of 12, and a gap penalty of 4 can beused. Yet another useful algorithm for identifying regions of localsequence similarity and alignment is the FASTA algorithm as described inPearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. Whenusing the FASTA algorithm for comparing nucleotide or amino acidsequences, a PAM120 weight residue table can, for example, be used witha k-tuple value of 2.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

The invention also provides chimeric or fusion proteins corresponding toa marker of the invention. As used herein, a “chimeric protein” or“fusion protein” comprises all or part (preferably a biologically activepart) of a polypeptide corresponding to a marker of the inventionoperably linked to a heterologous polypeptide (i.e., a polypeptide otherthan the polypeptide corresponding to the marker). Within the fusionprotein, the term “operably linked” is intended to indicate that thepolypeptide of the invention and the heterologous polypeptide are fusedin-frame to each other. The heterologous polypeptide can be fused to theamino-terminus or the carboxyl-terminus of the polypeptide of theinvention.

One useful fusion protein is a GST fusion protein in which a polypeptidecorresponding to a marker of the invention is fused to the carboxylterminus of GST sequences. Such fusion proteins can facilitate thepurification of a recombinant polypeptide of the invention.

In another embodiment, the fusion protein contains a heterologous signalsequence at its amino terminus. For example, the native signal sequenceof a polypeptide corresponding to a marker of the invention can beremoved and replaced with a signal sequence from another protein. Forexample, the gp67 secretory sequence of the baculovirus envelope proteincan be used as a heterologous signal sequence (Ausubel et al., ed.,Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1992).Other examples of eukaryotic heterologous signal sequences include thesecretory sequences of melittin and human placental alkaline phosphatase(Stratagene; La Jolla, Calif.). In yet another example, usefulprokaryotic heterologous signal sequences include the phoA secretorysignal (Sambrook et al., supra) and the protein A secretory signal(Pharmacia Biotech; Piscataway, N.J.).

In yet another embodiment, the fusion protein is an immunoglobulinfusion protein in which all or part of a polypeptide corresponding to amarker of the invention is fused to sequences derived from a member ofthe immunoglobulin protein family. The immunoglobulin fusion proteins ofthe invention can be incorporated into pharmaceutical compositions andadministered to a subject to inhibit an interaction between a ligand(soluble or membrane-bound) and a protein on the surface of a cell(receptor), to thereby suppress signal transduction in vivo. Theimmunoglobulin fusion protein can be used to affect the bioavailabilityof a cognate ligand of a polypeptide of the invention. Inhibition ofligand/receptor interaction can be useful therapeutically, both fortreating proliferative and differentiative disorders and for modulating(e.g. promoting or inhibiting) cell survival. Moreover, theimmunoglobulin fusion proteins of the invention can be used asimmunogens to produce antibodies directed against a polypeptide of theinvention in a subject, to purify ligands and in screening assays toidentify molecules which inhibit the interaction of receptors withligands.

Chimeric and fusion proteins of the invention can be produced bystandard recombinant DNA techniques. In another embodiment, the fusiongene can be synthesized by conventional techniques including automatedDNA synthesizers. Alternatively, PCR amplification of gene fragments canbe carried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (see,e.g., Ausubel et al., supra). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A nucleic acid encoding a polypeptide of the invention canbe cloned into such an expression vector such that the fusion moiety islinked in-frame to the polypeptide of the invention.

A signal sequence can be used to facilitate secretion and isolation ofthe secreted protein or other proteins of interest. Signal sequences aretypically characterized by a core of hydrophobic amino acids which aregenerally cleaved from the mature protein during secretion in one ormore cleavage events. Such signal peptides contain processing sites thatallow cleavage of the signal sequence from the mature proteins as theypass through the secretory pathway. Thus, the invention pertains to thedescribed polypeptides having a signal sequence, as well as topolypeptides from which the signal sequence has been proteolyticallycleaved (i.e., the cleavage products). In one embodiment, a nucleic acidsequence encoding a signal sequence can be operably linked in anexpression vector to a protein of interest, such as a protein which isordinarily not secreted or is otherwise difficult to isolate. The signalsequence directs secretion of the protein, such as from a eukaryotichost into which the expression vector is transformed, and the signalsequence is subsequently or concurrently cleaved. The protein can thenbe readily purified from the extracellular medium by art recognizedmethods. Alternatively, the signal sequence can be linked to the proteinof interest using a sequence which facilitates purification, such aswith a GST domain.

The present invention also pertains to variants of the polypeptidescorresponding to individual markers of the invention. Such variants havean altered amino acid sequence which can function as either agonists(mimetics) or as antagonists. Variants can be generated by mutagenesis,e.g., discrete point mutation or truncation. An agonist can retainsubstantially the same, or a subset, of the biological activities of thenaturally occurring form of the protein. An antagonist of a protein caninhibit one or more of the activities of the naturally occurring form ofthe protein by, for example, competitively binding to a downstream orupstream member of a cellular signaling cascade which includes theprotein of interest. Thus, specific biological effects can be elicitedby treatment with a variant of limited function. Treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein can have fewer side effects in asubject relative to treatment with the naturally occurring form of theprotein.

Variants of a protein of the invention which function as either agonists(mimetics) or as antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theprotein of the invention for agonist or antagonist activity. In oneembodiment, a variegated library of variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential protein sequences is expressible as individual polypeptides,or alternatively, as a set of larger fusion proteins (e.g., for phagedisplay). There are a variety of methods which can be used to producelibraries of potential variants of the polypeptides of the inventionfrom a degenerate oligonucleotide sequence. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang,1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem.53:323; Itakura et al., 1984, Science 198:1056; Ike et al., 1983 NucleicAcid Res. 11:477).

In addition, libraries of fragments of the coding sequence of apolypeptide corresponding to a marker of the invention can be used togenerate a variegated population of polypeptides for screening andsubsequent selection of variants. For example, a library of codingsequence fragments can be generated by treating a double stranded PCRfragment of the coding sequence of interest with a nuclease underconditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes amino terminal andinternal fragments of various sizes of the protein of interest.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high throughputanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofa protein of the invention (Arkin and Yourvan, 1992, Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al., 1993, Protein Engineering6(3):327-331).

An isolated polypeptide corresponding to a marker of the invention, or afragment thereof, can be used as an immunogen to generate antibodiesusing standard techniques for polyclonal and monoclonal antibodypreparation. The full-length polypeptide or protein can be used or,alternatively, the invention provides antigenic peptide fragments foruse as immunogens. The antigenic peptide of a protein of the inventioncomprises at least 8 (preferably 10, 15, 20, or 30 or more) amino acidresidues of the amino acid sequence of one of the polypeptides of theinvention, and encompasses an epitope of the protein such that anantibody raised against the peptide forms a specific immune complex witha marker of the invention to which the protein corresponds. Preferredepitopes encompassed by the antigenic peptide are regions that arelocated on the surface of the protein, e.g., hydrophilic regions.Hydrophobicity sequence analysis, hydrophilicity sequence analysis, orsimilar analyses can be used to identify hydrophilic regions.

An immunogen typically is used to prepare antibodies by immunizing asuitable (i.e. immunocompetent) subject such as a rabbit, goat, mouse,or other mammal or vertebrate. An appropriate immunogenic preparationcan contain, for example, recombinantly-expressed orchemically-synthesized polypeptide. The preparation can further includean adjuvant, such as Freund's complete or incomplete adjuvant, or asimilar immunostimulatory agent.

Accordingly, another aspect of the invention pertains to antibodiesdirected against a polypeptide of the invention. The terms “antibody”and “antibody substance” as used interchangeably herein refer toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds an antigen, such as a polypeptideof the invention. A molecule which specifically binds to a givenpolypeptide of the invention is a molecule which binds the polypeptide,but does not substantially bind other molecules in a sample, e.g., abiological sample, which naturally contains the polypeptide. Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)₂ fragments which can be generated by treating theantibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies. The term “monoclonal antibody” or“monoclonal antibody composition”, as used herein, refers to apopulation of antibody molecules that contain only one species of anantigen binding site capable of immunoreacting with a particularepitope.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a polypeptide of the invention as an immunogen.The antibody titer in the immunized subject can be monitored over timeby standard techniques, such as with an enzyme linked immunosorbentassay (ELISA) using immobilized polypeptide. If desired, the antibodymolecules can be harvested or isolated from the subject (e.g., from theblood or serum of the subject) and further purified by well-knowntechniques, such as protein A chromatography to obtain the IgG fraction.At an appropriate time after immunization, e.g., when the specificantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497, the human B cellhybridoma technique (see Kozbor et al., 1983, Immunol. Today 4:72), theEBV-hybridoma technique (see Cole et al., pp. 77-96 In MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., 1985) or triomatechniques. The technology for producing hybridomas is well known (seegenerally Current Protocols in Immunology, Coligan et al. ed., JohnWiley & Sons, New York, 1994). Hybridoma cells producing a monoclonalantibody of the invention are detected by screening the hybridomaculture supernatants for antibodies that bind the polypeptide ofinterest, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody directed against a polypeptide of the invention canbe identified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) withthe polypeptide of interest. Kits for generating and screening phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP Phage Display Kit, Catalog No. 240612). Additionally,examples of methods and reagents particularly amenable for use ingenerating and screening antibody display library can be found in, forexample, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCTPublication No. WO 91/17271; PCT Publication No. WO 92/20791; PCTPublication No. WO 92/15679; PCT Publication No. WO 93/01288; PCTPublication No. WO 92/01047; PCT Publication No. WO 92/09690; PCTPublication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J.12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in PCT Publication No. WO87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human subjects. Such antibodies can be produced usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide corresponding to a marker of the invention. Monoclonalantibodies directed against the antigen can be obtained usingconventional hybridoma technology. The human immunoglobulin transgenesharbored by the transgenic mice rearrange during B cell differentiation,and subsequently undergo class switching and somatic mutation. Thus,using such a technique, it is possible to produce therapeutically usefulIgG, IgA and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar (1995) Int. Rev.Immunol. 13:65-93). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., U.S. Pat. Nos. 5,625,126;5,633,425; 5,569,825; 5,661,016; and 5,545,806. In addition, companiessuch as Abgenix, Inc. (Freemont, Calif.), can be engaged to providehuman antibodies directed against a selected antigen using technologysimilar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al., 1994, Bio/technology12:899-903).

An antibody, antibody derivative, or fragment thereof, whichspecifically binds a marker of the invention which is overexpressed incancer (e.g., a marker set forth in Table 5), may be used to inhibitactivity of a marker, e.g., a marker set forth in Table 5, and thereforemay be administered to a subject to treat, inhibit, or prevent cancer inthe subject. Furthermore, conjugated antibodies may also be used totreat, inhibit, or prevent cancer in a subject. Conjugated antibodies,preferably monoclonal antibodies, or fragments thereof, are antibodieswhich are joined to drugs, toxins, or radioactive atoms, and used asdelivery vehicles to deliver those substances directly to cancer cells.The antibody, e.g., an antibody which specifically binds a marker of theinvention (e.g., a marker listed in Table 5), is administered to asubject and binds the marker, thereby delivering the toxic substance tothe cancer cell, minimizing damage to normal cells in other parts of thebody.

Conjugated antibodies are also referred to as “tagged,” “labeled,” or“loaded.” Antibodies with chemotherapeutic agents attached are generallyreferred to as chemolabeled. Antibodies with radioactive particlesattached are referred to as radiolabeled, and this type of therapy isknown as radioimmunotherapy (RIT). Aside from being used to treatcancer, radiolabeled antibodies can also be used to detect areas ofcancer spread in the body. Antibodies attached to toxins are calledimmunotoxins.

Immunotoxins are made by attaching toxins (e.g., poisonous substancesfrom plants or bacteria) to monoclonal antibodies. Immunotoxins may beproduced by attaching monoclonal antibodies to bacterial toxins such asdiphtherial toxin (DT) or pseudomonal exotoxin (PE40), or to planttoxins such as ricin A or saporin.

An antibody directed against a polypeptide corresponding to a marker ofthe invention (e.g., a monoclonal antibody) can be used to isolate thepolypeptide by standard techniques, such as affinity chromatography orimmunoprecipitation. Moreover, such an antibody can be used to detectthe marker (e.g., in a cellular lysate or cell supernatant) in order toevaluate the level and pattern of expression of the marker. Theantibodies can also be used diagnostically to monitor protein levels intissues or body fluids (e.g. in an ovary-associated body fluid) as partof a clinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

V. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptidecorresponding to a marker of the invention (or a portion of such apolypeptide). As used herein, the term “vector” refers to a to nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors, namely expressionvectors, are capable of directing the expression of genes to which theyare operably linked. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids (vectors).However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operably linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Methods in Enzymology: GeneExpression Technology vol. 185, Academic Press, San Diego, Calif.(1991). Regulatory sequences include those which direct constitutiveexpression of a nucleotide sequence in many types of host cell and thosewhich direct expression of the nucleotide sequence only in certain hostcells (e.g., tissue-specific regulatory sequences). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, andthe like. The expression vectors of the invention can be introduced intohost cells to thereby produce proteins or peptides, including fusionproteins or peptides, encoded by nucleic acids as described herein.

The recombinant expression vectors of the invention can be designed forexpression of a polypeptide corresponding to a marker of the inventionin prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells{using baculovirus expression vectors}, yeast cells or mammalian cells).Suitable host cells are discussed further in Goeddel, supra.Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d (Studieret al., p. 60-89, In Gene Expression Technology Methods in Enzymologyvol. 185, Academic Press, San Diego, Calif., 1991). Target geneexpression from the pTrc vector relies on host RNA polymerasetranscription from a hybrid trp-lac fusion promoter. Target geneexpression from the pET 11d vector relies on transcription from a T7gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase(T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) orHMS174(DE3) from a resident prophage harboring a T7 gn1 gene under thetranscriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, p. 119-128,In Gene Expression Technology: Methods in Enzymology vol. 185, AcademicPress, San Diego, Calif., 1990. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., 1992, Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kurjanand Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987,Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpPicZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, the expression vector is a baculovirus expression vector.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.,1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers, 1989, Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, 1987, Nature 329:840)and pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal., supra.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.,1987, Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton, 1988, Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) andimmunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen andBaltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989, Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al., 1985,Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss,1990, Science 249:374-379) and the α-fetoprotein promoter (Camper andTilghman, 1989, Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation: That is, the DNA molecule isoperably linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to the mRNA encoding a polypeptide of the invention.Regulatory sequences operably linked to a nucleic acid cloned in theantisense orientation can be chosen which direct the continuousexpression of the antisense RNA molecule in a variety of cell types, forinstance viral promoters and/or enhancers, or regulatory sequences canbe chosen which direct constitutive, tissue-specific or cell typespecific expression of antisense RNA. The antisense expression vectorcan be in the form of a recombinant plasmid, phagemid, or attenuatedvirus in which antisense nucleic acids are produced under the control ofa high efficiency regulatory region, the activity of which can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genessee Weintraub et al., 1986, Trends in Genetics, Vol. 1 (1).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell(e.g., insect cells, yeast or mammalian cells).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce a polypeptide corresponding to amarker of the invention. Accordingly, the invention further providesmethods for producing a polypeptide corresponding to a marker of theinvention using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding a polypeptide of the inventionhas been introduced) in a suitable medium such that the marker isproduced. In another embodiment, the method further comprises isolatingthe marker polypeptide from the medium or the host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichsequences encoding a polypeptide corresponding to a marker of theinvention have been introduced. Such host cells can then be used tocreate non-human transgenic animals in which exogenous sequencesencoding a marker protein of the invention have been introduced intotheir genome or homologous recombinant animals in which endogenousgene(s) encoding a polypeptide corresponding to a marker of theinvention sequences have been altered. Such animals are useful forstudying the function and/or activity of the polypeptide correspondingto the marker, for identifying and/or evaluating modulators ofpolypeptide activity, as well as in pre-clinical testing of therapeuticsor diagnostic molecules, for marker discovery or evaluation, e.g.,therapeutic and diagnostic marker discovery or evaluation, or assurrogates of drug efficacy and specificity.

As used herein, a “transgenic animal” is a non-human animal, preferablya mammal, more preferably a rodent such as a rat or mouse, in which oneor more of the cells of the animal includes a transgene. Other examplesof transgenic animals include non-human primates, sheep, dogs, cows,goats, chickens, amphibians, etc. A transgene is exogenous DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, an“homologous recombinant animal” is a non-human animal, preferably amammal, more preferably a mouse, in which an endogenous gene has beenaltered by homologous recombination between the endogenous gene and anexogenous DNA molecule introduced into a cell of the animal, e.g., anembryonic cell of the animal, prior to development of the animal.Transgenic animals also include inducible transgenic animals, such asthose described in, for example, Chan I. T., et al. (2004) J ClinInvest. 113(4):528-38 and Chin L. et al (1999) Nature 400(6743):468-72.

A transgenic animal of the invention can be created by introducing anucleic acid encoding a polypeptide corresponding to a marker of theinvention into the male pronuclei of a fertilized oocyte, e.g., bymicroinjection, retroviral infection, and allowing the oocyte to developin a pseudopregnant female foster animal. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the polypeptide of the invention toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan,Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1986. Similar methods are used for production ofother transgenic animals. A transgenic founder animal can be identifiedbased upon the presence of the transgene in its genome and/or expressionof mRNA encoding the transgene in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying thetransgene can further be bred to other transgenic animals carrying othertransgenes.

To create an homologous recombinant animal, a vector is prepared whichcontains at least a portion of a gene encoding a polypeptidecorresponding to a marker of the invention into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the gene. In a preferred embodiment, the vector isdesigned such that, upon homologous recombination, the endogenous geneis functionally disrupted (i.e., no longer encodes a functional protein;also referred to as a “knock out” vector). Alternatively, the vector canbe designed such that, upon homologous recombination, the endogenousgene is mutated or otherwise altered but still encodes functionalprotein (e.g., the upstream regulatory region can be altered to therebyalter the expression of the endogenous protein). In the homologousrecombination vector, the altered portion of the gene is flanked at its5′ and 3′ ends by additional nucleic acid of the gene to allow forhomologous recombination to occur between the exogenous gene carried bythe vector and an endogenous gene in an embryonic stem cell. Theadditional flanking nucleic acid sequences are of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector (see, e.g., Thomas and Capecchi, 1987, Cell51:503 for a description of homologous recombination vectors). Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced gene has homologouslyrecombined with the endogenous gene are selected (see, e.g., Li et al.,1992, Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see, e.g., Bradley, Teratocarcinomas and Embryonic Stem Cells: APractical Approach, Robertson, Ed., IRL, Oxford, 1987, pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination vectors and homologous recombinantanimals are described further in Bradley (1991) Current Opinion inBio/Technology 2:823-829 and in PCT Publication NOS. WO 90/11354, WO91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.,1991, Science 251:1351-1355). If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication NOS. WO 97/07668 and WO 97/07669.

VI. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject, e.g., a human, who has or is at risk of(or susceptible to) cancer, e.g., pancreatic cancer. As used herein,“treatment” of a subject includes the application or administration of atherapeutic agent to a subject, or application or administration of atherapeutic agent to a cell or tissue from a subject, who has a diseasesor disorder, has a symptom of a disease or disorder, or is at risk of(or susceptible to) a disease or disorder, with the purpose of curing,inhibiting, healing, alleviating, relieving, altering, remedying,ameliorating, improving, or affecting the disease or disorder, thesymptom of the disease or disorder, or the risk of (or susceptibilityto) the disease or disorder. As used herein, a “therapeutic agent” or“compound” includes, but is not limited to, small molecules, peptides,peptidomimetics, polypeptides, RNA interfering agents, e.g., siRNAmolecules, antibodies, ribozymes, and antisense oligonucleotides.

As described herein, cancer in subjects is associated with a change,e.g., an increase in the amount and/or activity, or a change in thestructure, of one or more markers listed in Table 5 (e.g., a marker thatwas shown to be increased in cancer), and/or a decrease in the amountand/or activity, or a change in the structure of one or more markerslisted in Table 4 (e.g. a marker that was shown to be decreased incancer). While, as discussed above, some of these changes in amount,structure, and/or activity, result from occurrence of the cancer, othersof these changes induce, maintain, and promote the cancerous state ofcancer, cells. Thus, cancer, characterized by an increase in the amountand/or activity, or a change in the structure, of one or more markerslisted in Table 5 (e.g., a marker that is shown to be increased incancer), can be inhibited by inhibiting amount, e.g., expression orprotein level, and/or activity of those markers. Likewise, cancercharacterized by a decrease in the amount and/or activity, or a changein the structure, of one or more markers listed in Table 4 (e.g., amarker that is shown to be decreased in cancer), can be inhibited byenhancing amount, e.g., expression or protein level, and/or activity ofthose markers.

Accordingly, another aspect of the invention pertains to methods fortreating a subject suffering from cancer. These methods involveadministering to a subject a compound which modulates amount and/oractivity of one or more markers of the invention. For example, methodsof treatment or prevention of cancer include administering to a subjecta compound which decreases the amount and/or activity of one or moremarkers listed in Table 5 (e.g., a marker that was shown to be increasedin cancer). Compounds, e.g., antagonists, which may be used to inhibitamount and/or activity of a marker listed in Table 5, to thereby treator prevent cancer include antibodies (e.g., conjugated antibodies),small molecules, RNA interfering agents, e.g., siRNA molecules,ribozymes, and antisense oligonucleotides. In one embodiment, anantibody used for treatment is conjugated to a toxin, a chemotherapeuticagent, or radioactive particles.

Methods of treatment or prevention of cancer also include administeringto a subject a compound which increases the amount and/or activity ofone or more markers listed in Table 4 (e.g., a marker that was shown tobe decreased in cancer). Compounds, e.g., agonists, which may be used toincrease expression or activity of a marker listed in Table 4, tothereby treat or prevent cancer include small molecules, peptides,peptoids, peptidomimetics, and polypeptides.

Small molecules used in the methods of the invention include those whichinhibit a protein-protein interaction and thereby either increase ordecrease marker amount and/or activity. Furthermore, modulators, e.g.,small molecules, which cause re-expression of silenced genes, e.g.,tumor suppressors, are also included herein. For example, such moleculesinclude compounds which interfere with DNA binding or methyltransferasactivity.

An aptamer may also be used to modulate, e.g., increase or inhibitexpression or activity of a marker of the invention to thereby treat,prevent or inhibit cancer. Aptamers are DNA or RNA molecules that havebeen selected from random pools based on their ability to bind othermolecules. Aptamers may be selected which bind nucleic acids orproteins.

VII. Screening Assays

The invention also provides methods (also referred to herein as“screening assays”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., proteins, peptides, peptidomimetics,peptoids, small molecules or other drugs) which (a) bind to the marker,or (b) have a modulatory (e.g., stimulatory or inhibitory) effect on theactivity of the marker or, more specifically, (c) have a modulatoryeffect on the interactions of the marker with one or more of its naturalsubstrates (e.g., peptide, protein, hormone, co-factor, or nucleicacid), or (d) have a modulatory effect on the expression of the marker.Such assays typically comprise a reaction between the marker and one ormore assay components. The other components may be either the testcompound itself, or a combination of test compound and a natural bindingpartner of the marker. Compounds identified via assays such as thosedescribed herein may be useful, for example, for modulating, e.g.,inhibiting, ameliorating, treating, or preventing cancer.

The test compounds of the present invention may be obtained from anyavailable source, including systematic libraries of natural and/orsynthetic compounds. Test compounds may also be obtained by any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann et al., 1994,J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam, 1997, AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994)J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/orspores, (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al, 1992,Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith, 1990,Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla-et al,1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol.222:301-310; Ladner, supra.).

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of a marker or biologicallyactive portion thereof. In another embodiment, the invention providesassays for screening candidate or test compounds which bind to a markeror biologically active portion thereof. Determining the ability of thetest compound to directly bind to a marker can be accomplished, forexample, by coupling the compound with a radioisotope or enzymatic labelsuch that binding of the compound to the marker can be determined bydetecting the labeled marker compound in a complex. For example,compounds (e.g., marker substrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemission or by scintillation counting.Alternatively, assay components can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product.

In another embodiment, the invention provides assays for screeningcandidate or test compounds which modulate the activity of a marker or abiologically active portion thereof. In all likelihood, the marker can,in vivo, interact with one or more molecules, such as, but not limitedto, peptides, proteins, hormones, cofactors and nucleic acids. For thepurposes of this discussion, such cellular and extracellular moleculesare referred to herein as “binding partners” or marker “substrate”.

One necessary embodiment of the invention in order to facilitate suchscreening is the use of the marker to identify its natural in vivobinding partners. There are many ways to accomplish this which are knownto one skilled in the art. One example is the use of the marker proteinas “bait protein” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al, 1993, Cell 72:223-232;Madura et al, 1993, J. Biol. Chem. 268:12046-12054; Bartel et al, 1993,Biotechniques 14:920-924; Iwabuchi et al, 1993 Oncogene 8:1693-1696;Brent WO94/10300) in order to identify other proteins which bind to orinteract with the marker (binding partners) and, therefore, are possiblyinvolved in the natural function of the marker. Such marker bindingpartners are also likely to be involved in the propagation of signals bythe marker or downstream elements of a marker-mediated signalingpathway. Alternatively, such marker binding partners may also be foundto be inhibitors of the marker.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that encodes a marker proteinfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a marker-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be readily detected and cell colonies containingthe functional transcription factor can be isolated and used to obtainthe cloned gene which encodes the protein which interacts with themarker protein.

In a further embodiment, assays may be devised through the use of theinvention for the purpose of identifying compounds which modulate (e.g.,affect either positively or negatively) interactions between a markerand its substrates and/or binding partners. Such compounds can include,but are not limited to, molecules such as antibodies, peptides,hormones, oligonucleotides, nucleic acids, and analogs thereof. Suchcompounds may also be obtained from any available source, includingsystematic libraries of natural and/or synthetic compounds. Thepreferred assay components for use in this embodiment is a cancer,marker identified herein, the known binding partner and/or substrate ofsame, and the test compound. Test compounds can be supplied from anysource.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the marker and its bindingpartner involves preparing a reaction mixture containing the marker andits binding partner under conditions and for a time sufficient to allowthe two products to interact and bind, thus forming a complex. In orderto test an agent for inhibitory activity, the reaction mixture isprepared in the presence and absence of the test compound. The testcompound can be initially included in the reaction mixture, or can beadded at a time subsequent to the addition of the marker and its bindingpartner. Control reaction mixtures are incubated without the testcompound or with a placebo. The formation of any complexes between themarker and its binding partner is then detected. The formation of acomplex in the control reaction, but less or no such formation in thereaction mixture containing the test compound, indicates that thecompound interferes with the interaction of the marker and its bindingpartner. Conversely, the formation of more complex in the presence ofcompound than in the control reaction indicates that the compound mayenhance interaction of the marker and its binding partner. The assay forcompounds that interfere with the interaction of the marker with itsbinding partner may be conducted in a heterogeneous or homogeneousformat. Heterogeneous assays involve anchoring either the marker or itsbinding partner onto a solid phase and detecting complexes anchored tothe solid phase at the end of the reaction. In homogeneous assays, theentire reaction is carried out in a liquid phase. In either approach,the order of addition of reactants can be varied to obtain differentinformation about the compounds being tested. For example, testcompounds that interfere with the interaction between the markers andthe binding partners (e.g., by competition) can be identified byconducting the reaction in the presence of the test substance, i.e., byadding the test substance to the reaction mixture prior to orsimultaneously with the marker and its interactive binding partner.Alternatively, test compounds that disrupt preformed complexes, e.g.compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction mixture after complexes have been formed. The variousformats are briefly described below.

In a heterogeneous assay system, either the marker or its bindingpartner is anchored onto a solid surface or matrix, while the othercorresponding non-anchored component may be labeled, either directly orindirectly. In practice, microtitre plates are often utilized for thisapproach. The anchored species can be immobilized by a number ofmethods, either non-covalent or covalent, that are typically well knownto one who practices the art. Non-covalent attachment can often beaccomplished simply by coating the solid surface with a solution of themarker or its binding partner and drying. Alternatively, an immobilizedantibody specific for the assay component to be anchored can be used forthis purpose. Such surfaces can often be prepared in advance and stored.

In related embodiments, a fusion protein can be provided which adds adomain that allows one or both of the assay components to be anchored toa matrix. For example, glutathione-S-transferase/marker fusion proteinsor glutathione-S-transferase/binding partner can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedmarker or its binding partner, and the mixture incubated underconditions conducive to complex formation (e.g., physiologicalconditions). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound assay components, the immobilizedcomplex assessed either directly or indirectly, for example, asdescribed above. Alternatively, the complexes can be dissociated fromthe matrix, and the level of marker binding or activity determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a markeror a marker binding partner can be immobilized utilizing conjugation ofbiotin and streptavidin. Biotinylated marker protein or target moleculescan be prepared from biotin-NHS(N-hydroxy-succinimide) using techniquesknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). In certain embodiments, theprotein-immobilized surfaces can be prepared in advance and stored.

In order to conduct the assay, the corresponding partner of theimmobilized assay component is exposed to the coated surface with orwithout the test compound. After the reaction is complete, unreactedassay components are removed (e.g., by washing) and any complexes formedwill remain immobilized on the solid surface. The detection of complexesanchored on the solid surface can be accomplished in a number of ways.Where the non-immobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the initially non-immobilizedspecies (the antibody, in turn, can be directly labeled or indirectlylabeled with, e.g., a labeled anti-Ig antibody). Depending upon theorder of addition of reaction components, test compounds which modulate(inhibit or enhance) complex formation or which disrupt preformedcomplexes can be detected.

In an alternate embodiment of the invention, a homogeneous assay may beused. This is typically a reaction, analogous to those mentioned above,which is conducted in a liquid phase in the presence or absence of thetest compound. The formed complexes are then separated from unreactedcomponents, and the amount of complex formed is determined. As mentionedfor heterogeneous assay systems, the order of addition of reactants tothe liquid phase can yield information about which test compoundsmodulate (inhibit or enhance) complex formation and which disruptpreformed complexes.

In such a homogeneous assay, the reaction products may be separated fromunreacted assay components by any of a number of standard techniques,including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, complexes of molecules may be separated from uncomplexedmolecules through a series of centrifugal steps, due to the differentsedimentation equilibria of complexes based on their different sizes anddensities (see, for example, Rivas, G., and Minton, A. P., TrendsBiochem Sci 1993 August; 18(8):284-7). Standard chromatographictechniques may also be utilized to separate complexed molecules fromuncomplexed ones. For example, gel filtration chromatography separatesmolecules based on size, and through the utilization of an appropriategel filtration resin in a column format, for example, the relativelylarger complex may be separated from the relatively smaller uncomplexedcomponents. Similarly, the relatively different charge properties of thecomplex as compared to the uncomplexed molecules may be exploited todifferentially separate the complex from the remaining individualreactants, for example through the use of ion-exchange chromatographyresins. Such resins and chromatographic techniques are well known to oneskilled in the art (see, e.g., Heegaard, 1998, J. Mol. Recognit.11:141-148; Hage and Tweed, 1997, J. Chromatogr. B. Biomed. Sci. Appl.,699:499-525). Gel electrophoresis may also be employed to separatecomplexed molecules from unbound species (see, e.g., Ausubel et al(eds.), In: Current Protocols in Molecular Biology, J. Wiley & Sons, NewYork. 1999). In this technique, protein or nucleic acid complexes areseparated based on size or charge, for example. In order to maintain thebinding interaction during the electrophoretic process, nondenaturinggels in the absence of reducing agent are typically preferred, butconditions appropriate to the particular interactants will be well knownto one skilled in the art. Immunoprecipitation is another commontechnique utilized for the isolation of a protein-protein complex fromsolution (see, e.g., Ausubel et al (eds.), In: Current Protocols inMolecular Biology, J. Wiley & Sons, New York. 1999). In this technique,all proteins binding to an antibody specific to one of the bindingmolecules are precipitated from solution by conjugating the antibody toa polymer bead that may be readily collected by centrifugation. Thebound assay components are released from the beads (through a specificproteolysis event or other technique well known in the art which willnot disturb the protein-protein interaction in the complex), and asecond immunoprecipitation step is performed, this time utilizingantibodies specific for the correspondingly different interacting assaycomponent. In this manner, only formed complexes should remain attachedto the beads. Variations in complex formation in both the presence andthe absence of a test compound can be compared, thus offeringinformation about the ability of the compound to modulate interactionsbetween the marker and its binding partner.

Also within the scope of the present invention are methods for directdetection of interactions between the marker and its natural bindingpartner and/or a test compound in a homogeneous or heterogeneous assaysystem without further sample manipulation. For example, the techniqueof fluorescence energy transfer may be utilized (see, e.g., Lakowicz etal, U.S. Pat. No. 5,631,169; Stavrianopoulos et al, U.S. Pat. No.4,868,103). Generally, this technique involves the addition of afluorophore label on a first ‘donor’ molecule (e.g., marker or testcompound) such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule (e.g., marker or testcompound), which in turn is able to fluoresce due to the absorbedenergy. Alternately, the ‘donor’ protein molecule may simply utilize thenatural fluorescent energy of tryptophan residues. Labels are chosenthat emit different wavelengths of light, such that the ‘acceptor’molecule label may be differentiated from that of the ‘donor’. Since theefficiency of energy transfer between the labels is related to thedistance separating the molecules, spatial relationships between themolecules can be assessed. In a situation in which binding occursbetween the molecules, the fluorescent emission of the ‘acceptor’molecule label in the assay should be maximal. An FET binding event canbe conveniently measured through standard fluorometric detection meanswell known in the art (e.g., using a fluorimeter). A test substancewhich either enhances or hinders participation of one of the species inthe preformed complex will result in the generation of a signal variantto that of background. In this way, test substances that modulateinteractions between a marker and its binding partner can be identifiedin controlled assays.

In another embodiment, modulators of marker expression are identified ina method wherein a cell is contacted with a candidate compound and theexpression of mRNA or protein, corresponding to a marker in the cell, isdetermined. The level of expression of mRNA or protein in the presenceof the candidate compound is compared to the level of expression of mRNAor protein in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of marker expressionbased on this comparison. For example, when expression of marker mRNA orprotein is greater (statistically significantly greater) in the presenceof the candidate compound than in its absence, the candidate compound isidentified as a stimulator of marker mRNA or protein expression.Conversely, when expression of marker mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of marker mRNA or protein expression. The level of marker mRNAor protein expression in the cells can be determined by methodsdescribed herein for detecting marker mRNA or protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a marker protein can be furtherconfirmed in vivo, e.g., in a whole animal model for cancer, cellulartransformation and/or tumorigenesis. An animal model for pancreaticcancer is described in, for example, Aguirre A., et al. (2003) GenesDev. December 15; 17(24):3112-26, the contents of which are expresslyincorporated herein by reference. Additional animal based models ofcancer are well known in the art (reviewed in Animal Models of CancerPredisposition Syndromes, Hiai, H and Hino, O (eds.) 1999, Progress inExperimental Tumor Research, Vol. 35; Clarke A R Carcinogenesis (2000)21:435-41) and include, for example, carcinogen-induced tumors(Rithidech, K et al. Mutat Res (1999) 428:33-39; Miller, M L et al.Environ Mol Mutagen (2000) 35:319-327), injection and/or transplantationof tumor cells into an animal, as well as animals bearing mutations ingrowth regulatory genes, for example, oncogenes (e.g., ras) (Arbeit, J Met al. Am J Pathol (1993) 142:1187-1197; Sinn, E et al. Cell (1987)49:465-475; Thorgeirsson, S S et al. Toxicol Lett (2000)112-113:553-555) and tumor suppressor genes (e.g., p53) (Vooijs, M etal. Oncogene (1999) 18:5293-5303; Clark A R Cancer Metast Rev (1995)14:125-148; Kumar, T R et al. J Intern Med (1995) 238:233-238;Donehower, L A et al. (1992) Nature 356215-221). Furthermore,experimental model systems are available for the study of, for example,ovarian cancer (Hamilton, T C et al. Semin Oncol (1984) 11:285-298;Rahman, N A et al. Mol Cell Endocrinol (1998) 145:167-174; Beamer, W Get al. Toxicol Pathol (1998) 26:704-710), gastric cancer (Thompson, J etal. Int J Cancer (2000) 86:863-869; Fodde, R et al. Cytogenet Cell Genet(1999) 86:105-111), breast cancer (Li, M et al. Oncogene (2000)19:1010-1019; Green, J E et al. Oncogene (2000) 19:1020-1027), melanoma(Satyamoorthy, K et al. Cancer Metast Rev (1999) 18:401-405), andprostate cancer (Shirai, T et al. Mutat Res (2000) 462:219-226;Bostwick, D G et al. Prostate (2000) 43:286-294). Animal modelsdescribed in, for example, Chin L. et al (1999) Nature 400(6743):468-72,may also be used in the methods of the invention.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a marker modulating agent, a small molecule, anantisense marker nucleic acid molecule, a ribozyme, a marker-specificantibody, or fragment thereof, a marker protein, a marker nucleic acidmolecule, an RNA interfering agent, e.g., an siRNA molecule targeting amarker of the invention, or a marker-binding partner) can be used in ananimal model to determine the efficacy, toxicity, or side effects oftreatment with such an agent. Alternatively, an agent identified asdescribed herein can be used in an animal model to determine themechanism of action of such an agent. Furthermore, this inventionpertains to uses of novel agents identified by the above-describedscreening assays for treatments as described herein.

VIII. Pharmaceutical Compositions

The small molecules, peptides, peptoids, peptidomimetics, polypeptides,RNA interfering agents, e.g., siRNA molecules, antibodies, ribozymes,and antisense oligonucleotides (also referred to herein as “activecompounds” or “compounds”) corresponding to a marker of the inventioncan be incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the smallmolecules, peptides, peptoids, peptidomimetics, polypeptides, RNAinterfering agents, e.g., siRNA molecules, antibodies, ribozymes, orantisense oligonucleotides and a pharmaceutically acceptable carrier. Asused herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

The invention includes methods for preparing pharmaceutical compositionsfor modulating the expression or activity of a polypeptide or nucleicacid corresponding to a marker of the invention. Such methods compriseformulating a pharmaceutically acceptable carrier with an agent whichmodulates expression or activity of a polypeptide or nucleic acidcorresponding to a marker of the invention. Such compositions canfurther include additional active agents. Thus, the invention furtherincludes methods for preparing a pharmaceutical composition byformulating a pharmaceutically acceptable carrier with an agent whichmodulates expression or activity of a polypeptide or nucleic acidcorresponding to a marker of the invention and one or more additionalactive compounds.

It is understood that appropriate doses of small molecule agents andprotein or polypeptide agents depends upon a number of factors withinthe knowledge of the ordinarily skilled physician, veterinarian, orresearcher. The dose(s) of these agents will vary, for example,depending upon the identity, size, and condition of the subject orsample being treated, further depending upon the route by which thecomposition is to be administered, if applicable, and the effect whichthe practitioner desires the agent to have upon the nucleic acidmolecule or polypeptide of the invention. Small molecules include, butare not limited to, peptides, peptidomimetics, amino acids, amino acidanalogs, polynucleotides, polynucleotide analogs, nucleotides,nucleotide analogs, organic or inorganic compounds (i.e., includingheteroorganic and organometallic compounds) having a molecular weightless than about 10,000 grams per mole, organic or inorganic compoundshaving a molecular weight less than about 5,000 grams per mole, organicor inorganic compounds having a molecular weight less than about 1,000grams per mole, organic or inorganic compounds having a molecular weightless than about 500 grams per mole, and salts, esters, and otherpharmaceutically acceptable forms of such compounds.

Exemplary doses of a small molecule include milligram or microgramamounts per kilogram of subject or sample weight (e.g. about 1 microgramper kilogram to about 500 milligrams per kilogram, about 100 microgramsper kilogram to about 5 milligrams per kilogram, or about 1 microgramper kilogram to about 50 micrograms per kilogram).

As defined herein, a therapeutically effective amount of an RNAinterfering agent, e.g., siRNA, (i.e., an effective dosage) ranges fromabout 0.001 to 3,000 mg/kg body weight, preferably about 0.01 to 2500mg/kg body weight, more preferably about 0.1 to 2000, about 0.1 to 1000mg/kg body weight, 0.1 to 500 mg/kg body weight, 0.1 to 100 mg/kg bodyweight, 0.1 to 50 mg/kg body weight, 0.1 to 25 mg/kg body weight, andeven more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4to 7 mg/kg, or 5 to 6 mg/kg body weight. Treatment of a subject with atherapeutically effective amount of an RNA interfering agent can includea single treatment or, preferably, can include a series of treatments.In a preferred example, a subject is treated with an RNA interferingagent in the range of between about 0.1 to 20 mg/kg body weight, onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks.

Exemplary doses of a protein or polypeptide include gram, milligram ormicrogram amounts per kilogram of subject or sample weight (e.g. about 1microgram per kilogram to about 5 grams per kilogram, about 100micrograms per kilogram to about 500 milligrams per kilogram, or about 1milligram per kilogram to about 50 milligrams per kilogram). It isfurthermore understood that appropriate doses of one of these agentsdepend upon the potency of the agent with respect to the expression oractivity to be modulated. Such appropriate doses can be determined usingthe assays described herein. When one or more of these agents is to beadministered to an animal (e.g. a human) in order to modulate expressionor activity of a polypeptide or nucleic acid of the invention, aphysician, veterinarian, or researcher can, for example, prescribe arelatively low dose at first, subsequently increasing the dose until anappropriate response is obtained. In addition, it is understood that thespecific dose level for any particular animal subject will depend upon avariety of factors including the activity of the specific agentemployed, the age, body weight, general health, gender, and diet of thesubject, the time of administration, the route of administration, therate of excretion, any drug combination, and the degree of expression oractivity to be modulated.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediamine-tetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a polypeptide or antibody) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium, and thenincorporating the required other ingredients from those enumeratedabove. In the case of sterile powders for the preparation of sterileinjectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches, and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g. a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes having monoclonal antibodies incorporated thereinor thereon) can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of bodyweight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act inthe brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into theepithelium). A method for lipidation of antibodies is described byCruikshank et al. (1997) J Acquired Immune Deficiency Syndromes andHuman Retrovirology 14:193.

The nucleic acid molecules corresponding to a marker of the inventioncan be inserted into vectors and used as gene therapy vectors. Genetherapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (U.S. Pat. No. 5,328,470),or by stereotactic injection (see, e.g., Chen et al., 1994, Proc. Natl.Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the genetherapy vector can include the gene therapy vector in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery vector can be produced intact from recombinant cells, e.g.retroviral vectors, the pharmaceutical preparation can include one ormore cells which produce the gene delivery system.

The RNA interfering agents, e.g., siRNAs used in the methods of theinvention can be inserted into vectors. These constructs can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the vector can includethe RNA interfering agent, e.g., the siRNA vector in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery vector can be produced intact from recombinant cells, e.g.,retroviral vectors, the pharmaceutical preparation can include one ormore cells which produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

IX. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningthe amount, structure, and/or activity of polypeptides or nucleic acidscorresponding to one or more markers of the invention, in order todetermine whether an individual is at risk of developing cancer. Suchassays can be used for prognostic or predictive purposes to therebyprophylactically treat an individual prior to the onset of the cancer.

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs or other compounds administered either to inhibitcancer, or to treat or prevent any other disorder {i.e. in order tounderstand any carcinogenic effects that such treatment may have}) onthe amount, structure, and/or activity of a marker of the invention inclinical trials. These and other agents are described in further detailin the following sections.

A. Diagnostic Assays

1. Methods for Detection of Copy Number

Methods of evaluating the copy number of a particular marker orchromosomal region (e.g., an MCR) are well known to those of skill inthe art. The presence or absence of chromosomal gain or loss can beevaluated simply by a determination of copy number of the regions ormarkers identified herein.

Methods for evaluating copy number of encoding nucleic acid in a sampleinclude, but are not limited to, hybridization-based assays. Forexample, one method for evaluating the copy number of encoding nucleicacid in a sample involves a Southern Blot. In a Southern Blot, thegenomic DNA (typically fragmented and separated on an electrophoreticgel) is hybridized to a probe specific for the target region. Comparisonof the intensity of the hybridization signal from the probe for thetarget region with control probe signal from analysis of normal genomicDNA (e.g., a non-amplified portion of the same or related cell, tissue,organ, etc.) provides an estimate of the relative copy number of thetarget nucleic acid.

An alternative means for determining the copy number is in situhybridization (e.g., Angerer (1987) Meth. Enzymol 152: 649). Generally,in situ hybridization comprises the following steps: (1) fixation oftissue or biological structure to be analyzed; (2) prehybridizationtreatment of the biological structure to increase accessibility oftarget DNA, and to reduce nonspecific binding; (3) hybridization of themixture of nucleic acids to the nucleic acid in the biological structureor tissue; (4) post-hybridization washes to remove nucleic acidfragments not bound in the hybridization and (5) detection of thehybridized nucleic acid fragments. The reagent used in each of thesesteps and the conditions for use vary depending on the particularapplication.

Preferred hybridization-based assays include, but are not limited to,traditional “direct probe” methods such as Southern blots or in situhybridization (e.g., FISH), and “comparative probe” methods such ascomparative genomic hybridization (CGH), e.g., cDNA-based oroligonucleotide-based CGH. The methods can be used in a wide variety offormats including, but not limited to, substrate (e.g. membrane orglass) bound methods or array-based approaches.

In a typical in situ hybridization assay, cells are fixed to a solidsupport, typically a glass slide. If a nucleic acid is to be probed, thecells are typically denatured with heat or alkali. The cells are thencontacted with a hybridization solution at a moderate temperature topermit annealing of labeled probes specific to the nucleic acid sequenceencoding the protein. The targets (e.g., cells) are then typicallywashed at a predetermined stringency or at an increasing stringencyuntil an appropriate signal to noise ratio is obtained.

The probes are typically labeled, e.g., with radioisotopes orfluorescent reporters. Preferred probes are sufficiently long so as tospecifically hybridize with the target nucleic acid(s) under stringentconditions. The preferred size range is from about 200 bases to about1000 bases.

In some applications it is necessary to block the hybridization capacityof repetitive sequences. Thus, in some embodiments, tRNA, human genomicDNA, or Cot-I DNA is used to block non-specific hybridization.

In CGH methods, a first collection of nucleic acids (e.g. from a sample,e.g., a possible tumor) is labeled with a first label, while a secondcollection of nucleic acids (e.g. a control, e.g., from a healthycell/tissue) is labeled with a second label. The ratio of hybridizationof the nucleic acids is determined by the ratio of the two (first andsecond) labels binding to each fiber in the array. Where there arechromosomal deletions or multiplications, differences in the ratio ofthe signals from the two labels will be detected and the ratio willprovide a measure of the copy number. Array-based CGH may also beperformed with single-color labeling (as opposed to labeling the controland the possible tumor sample with two different dyes and mixing themprior to hybridization, which will yield ratio due to competitivehybridization to probes on the arrays). In single color CGH, the controlis labeled and hybridized to one array and absolute signals are read,and the possible tumor sample is labeled and hybridized to a secondarray (with identical content) and absolute signals are read. Copynumber difference is calculated based on absolute signals from the twoarrays. Hybridization protocols suitable for use with the methods of theinvention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234;Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No.430, 402; Methods in Molecular Biology, Vol. 33: In Situ HybridizationProtocols, Choo, ed., Humana Press, Totowa, N.J. (1994), etc. In oneembodiment, the hybridization protocol of Pinkel et al. (1998) NatureGenetics 20: 207-211, or of Kallioniemi (1992) Proc. Natl Acad Sci USA89:5321-5325 (1992) is used.

The methods of the invention are particularly well suited to array-basedhybridization formats. Array-based CGH is described in U.S. Pat. No.6,455,258, the contents of which are incorporated herein by reference.

In still another embodiment, amplification-based assays can be used tomeasure copy number. In such amplification-based assays, the nucleicacid sequences act as a template in an amplification reaction (e.g.,Polymerase Chain Reaction (PCR). In a quantitative amplification, theamount of amplification product will be proportional to the amount oftemplate in the original sample. Comparison to appropriate controls,e.g. healthy tissue, provides a measure of the copy number.

Methods of “quantitative” amplification are well known to those of skillin the art. For example, quantitative PCR involves simultaneouslyco-amplifying a known quantity of a control sequence using the sameprimers. This provides an internal standard that may be used tocalibrate the PCR reaction. Detailed protocols for quantitative PCR areprovided in Innis et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc. N.Y.). Measurement of DNA copy numberat microsatellite loci using quantitative PCR analysis is described inGinzonger, et al (2000) Cancer Research 60:5405-5409. The known nucleicacid sequence for the genes is sufficient to enable one of skill in theart to routinely select primers to amplify any portion of the gene.Fluorogenic quantitative PCR may also be used in the methods of theinvention. In fluorogenic quantitative PCR, quantitation is based onamount of fluorescence signals, e.g., TaqMan and sybr green.

Other suitable amplification methods include, but are not limited to,ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560,Landegren et al. (1988) Science 241: 1077, and Barringer et al. (1990)Gene 89: 117, transcription amplification (Kwoh et al. (1989) Proc.Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication(Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR,and linker adapter PCR, etc.

Loss of heterozygosity (LOH) mapping (Wang Z. C. et al. (2004) CancerRes 64(1):64-71; Seymour, A. B., et al. (1994) Cancer Res 54, 2761-4;Hahn, S. A., et al. (1995) Cancer Res 55, 4670-5; Kimura, M., et al.(1996) Genes Chromosomes Cancer 17, 88-93) may also be used to identifyregions of amplification or deletion.

2. Methods for Detection of Gene Expression

Marker expression level can also be assayed as a method for diagnosis ofcancer or risk for developing cancer. Expression of a marker of theinvention may be assessed by any of a wide variety of well known methodsfor detecting expression of a transcribed molecule or protein.Non-limiting examples of such methods include immunological methods fordetection of secreted, cell-surface, cytoplasmic, or nuclear proteins,protein purification methods, protein function or activity assays,nucleic acid hybridization methods, nucleic acid reverse transcriptionmethods, and nucleic acid amplification methods.

In preferred embodiments, activity of a particular gene is characterizedby a measure of gene transcript (e.g. mRNA), by a measure of thequantity of translated protein, or by a measure of gene productactivity. Marker expression can be monitored in a variety of ways,including by detecting mRNA levels, protein levels, or protein activity,any of which can be measured using standard techniques. Detection caninvolve quantification of the level of gene expression (e.g., genomicDNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can bea qualitative assessment of the level of gene expression, in particularin comparison with a control level. The type of level being detectedwill be clear from the context.

Methods of detecting and/or quantifying the gene transcript (mRNA orcDNA made therefrom) using nucleic acid hybridization techniques areknown to those of skill in the art (see Sambrook et al. supra). Forexample, one method for evaluating the presence, absence, or quantity ofcDNA involves a Southern transfer as described above. Briefly, the mRNAis isolated (e.g. using an acid guanidinium-phenol-chloroform extractionmethod, Sambrook et al. supra.) and reverse transcribed to produce cDNA.The cDNA is then optionally digested and run on a gel in buffer andtransferred to membranes. Hybridization is then carried out using thenucleic acid probes specific for the target cDNA.

A general principle of such diagnostic and prognostic assays involvespreparing a sample or reaction mixture that may contain a marker, and aprobe, under appropriate conditions and for a time sufficient to allowthe marker and probe to interact and bind, thus forming a complex thatcan be removed and/or detected in the reaction mixture. These assays canbe conducted in a variety of ways.

For example, one method to conduct such an assay would involve anchoringthe marker or probe onto a solid phase support, also referred to as asubstrate, and detecting target marker/probe complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, a sample from a subject, which is to be assayed for presenceand/or concentration of marker, can be anchored onto a carrier or solidphase support. In another embodiment, the reverse situation is possible,in which the probe can be anchored to a solid phase and a sample from asubject can be allowed to react as an unanchored component of the assay.

There are many established methods for anchoring assay components to asolid phase. These include, without limitation, marker or probemolecules which are immobilized through conjugation of biotin andstreptavidin. Such biotinylated assay components can be prepared frombiotin-NHS(N-hydroxy-succinimide) using techniques known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). In certain embodiments, the surfaces with immobilized assaycomponents can be prepared in advance and stored.

Other suitable carriers or solid phase supports for such assays includeany material capable of binding the class of molecule to which themarker or probe belongs. Well-known supports or carriers include, butare not limited to, glass, polystyrene, nylon, polypropylene, nylon,polyethylene, dextran, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite.

In order to conduct assays with the above mentioned approaches, thenon-immobilized component is added to the solid phase upon which thesecond component is anchored. After the reaction is complete,uncomplexed components may be removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized uponthe solid phase. The detection of marker/probe complexes anchored to thesolid phase can be accomplished in a number of methods outlined herein.

In a preferred embodiment, the probe, when it is the unanchored assaycomponent, can be labeled for the purpose of detection and readout ofthe assay, either directly or indirectly, with detectable labelsdiscussed herein and which are well-known to one skilled in the art.

It is also possible to directly detect marker/probe complex formationwithout further manipulation or labeling of either component (marker orprobe), for example by utilizing the technique of fluorescence energytransfer (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169;Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore labelon the first, ‘donor’ molecule is selected such that, upon excitationwith incident light of appropriate wavelength, its emitted fluorescentenergy will be absorbed by a fluorescent label on a second ‘acceptor’molecule, which in turn is able to fluoresce due to the absorbed energy.Alternately, the ‘donor’ protein molecule may simply utilize the naturalfluorescent energy of tryptophan residues. Labels are chosen that emitdifferent wavelengths of light, such that the ‘acceptor’ molecule labelmay be differentiated from that of the ‘donor’. Since the efficiency ofenergy transfer between the labels is related to the distance separatingthe molecules, spatial relationships between the molecules can beassessed. In a situation in which binding occurs between the molecules,the fluorescent emission of the ‘acceptor’ molecule label in the assayshould be maximal. An FET binding event can be conveniently measuredthrough standard fluorometric detection means well known in the art(e.g., using a fluorimeter).

In another embodiment, determination of the ability of a probe torecognize a marker can be accomplished without labeling either assaycomponent (probe or marker) by utilizing a technology such as real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S, andUrbaniczky, C., 1991, Anal. Chem. 63:2338-2345 and Szabo et al., 1995,Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” or “surfaceplasmon resonance” is a technology for studying biospecific interactionsin real time, without labeling any of the interactants (e.g., BIAcore).Changes in the mass at the binding surface (indicative of a bindingevent) result in alterations of the refractive index of light near thesurface (the optical phenomenon of surface plasmon resonance (SPR)),resulting in a detectable signal which can be used as an indication ofreal-time reactions between biological molecules.

Alternatively, in another embodiment, analogous diagnostic andprognostic assays can be conducted with marker and probe as solutes in aliquid phase. In such an assay, the complexed marker and probe areseparated from uncomplexed components by any of a number of standardtechniques, including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, marker/probe complexes may be separated from uncomplexedassay components through a series of centrifugal steps, due to thedifferent sedimentation equilibria of complexes based on their differentsizes and densities (see, for example, Rivas, G., and Minton, A. P.,1993, Trends Biochem Sci. 18(8):284-7). Standard chromatographictechniques may also be utilized to separate complexed molecules fromuncomplexed ones. For example, gel filtration chromatography separatesmolecules based on size, and through the utilization of an appropriategel filtration resin in a column format, for example, the relativelylarger complex may be separated from the relatively smaller uncomplexedcomponents. Similarly, the relatively different charge properties of themarker/probe complex as compared to the uncomplexed components may beexploited to differentiate the complex from uncomplexed components, forexample through the utilization of ion-exchange chromatography resins.Such resins and chromatographic techniques are well known to one skilledin the art (see, e.g., Heegaard, N. H., 1998, J. Mol. Recognit. Winter11 (1-6):141-8; Hage, D. S., and Tweed, S. A. J Chromatogr B Biomed SciAppl 1997 Oct. 10; 699 (1-2):499-525). Gel electrophoresis may also beemployed to separate complexed assay components from unbound components(see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology,John Wiley & Sons, New York, 1987-1999). In this technique, protein ornucleic acid complexes are separated based on size or charge, forexample. In order to maintain the binding interaction during theelectrophoretic process, non-denaturing gel matrix materials andconditions in the absence of reducing agent are typically preferred.Appropriate conditions to the particular assay and components thereofwill be well known to one skilled in the art.

In a particular embodiment, the level of mRNA corresponding to themarker can be determined both by in situ and by in vitro formats in abiological sample using methods known in the art. The term “biologicalsample” is intended to include tissues, cells, biological fluids andisolates thereof, isolated from a subject, as well as tissues, cells andfluids present within a subject. Many expression detection methods useisolated RNA. For in vitro methods, any RNA isolation technique thatdoes not select against the isolation of mRNA can be utilized for thepurification of RNA from cells (see, e.g., Ausubel et al., ed., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York 1987-1999).Additionally, large numbers of tissue samples can readily be processedusing techniques well known to those of skill in the art, such as, forexample, the single-step RNA isolation process of Chomczynski (1989,U.S. Pat. No. 4,843,155).

The isolated nucleic acid can be used in hybridization or amplificationassays that include, but are not limited to, Southern or Northernanalyses, polymerase chain reaction analyses and probe arrays. Onepreferred diagnostic method for the detection of mRNA levels involvescontacting the isolated mRNA with a nucleic acid molecule (probe) thatcan hybridize to the mRNA encoded by the gene being detected. Thenucleic acid probe can be, for example, a full-length cDNA, or a portionthereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250or 500 nucleotides in length and sufficient to specifically hybridizeunder stringent conditions to a mRNA or genomic DNA encoding a marker ofthe present invention. Other suitable probes for use in the diagnosticassays of the invention are described herein. Hybridization of an mRNAwith the probe indicates that the marker in question is being expressed.

In one format, the mRNA is immobilized on a solid surface and contactedwith a probe, for example by running the isolated mRNA on an agarose geland transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probe(s) are immobilizedon a solid surface and the mRNA is contacted with the probe(s), forexample, in an Affymetrix gene chip array. A skilled artisan can readilyadapt known mRNA detection methods for use in detecting the level ofmRNA encoded by the markers of the present invention.

The probes can be full length or less than the full length of thenucleic acid sequence encoding the protein. Shorter probes areempirically tested for specificity. Preferably nucleic acid probes are20 bases or longer in length. (See, e.g., Sambrook et al. for methods ofselecting nucleic acid probe sequences for use in nucleic acidhybridization.) Visualization of the hybridized portions allows thequalitative determination of the presence or absence of cDNA.

An alternative method for determining the level of a transcriptcorresponding to a marker of the present invention in a sample involvesthe process of nucleic acid amplification, e.g., by rtPCR (theexperimental embodiment set forth in Mullis, 1987, U.S. Pat. No.4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci.USA, 88:189-193), self sustained sequence replication (Guatelli et al.,1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptionalamplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No.5,854,033) or any other nucleic acid amplification method, followed bythe detection of the amplified molecules using techniques well known tothose of skill in the art. Fluorogenic rtPCR may also be used in themethods of the invention. In fluorogenic rtPCR, quantitation is based onamount of fluorescence signals, e.g., TaqMan and sybr green. Thesedetection schemes are especially useful for the detection of nucleicacid molecules if such molecules are present in very low numbers. Asused herein, amplification primers are defined as being a pair ofnucleic acid molecules that can anneal to 5′ or 3′ regions of a gene(plus and minus strands, respectively, or vice-versa) and contain ashort region in between. In general, amplification primers are fromabout 10 to 30 nucleotides in length and flank a region from about 50 to200 nucleotides in length. Under appropriate conditions and withappropriate reagents, such primers permit the amplification of a nucleicacid molecule comprising the nucleotide sequence flanked by the primers.For in situ methods, mRNA does not need to be isolated from the cellsprior to detection. In such methods, a cell or tissue sample isprepared/processed using known histological methods. The sample is thenimmobilized on a support, typically a glass slide, and then contactedwith a probe that can hybridize to mRNA that encodes the marker.

As an alternative to making determinations based on the absoluteexpression level of the marker, determinations may be based on thenormalized expression level of the marker. Expression levels arenormalized by correcting the absolute expression level of a marker bycomparing its expression to the expression of a gene that is not amarker, e.g., a housekeeping gene that is constitutively expressed.Suitable genes for normalization include housekeeping genes such as theactin gene, or epithelial cell-specific genes. This normalization allowsthe comparison of the expression level in one sample, e.g., a subjectsample, to another sample, e.g., a non-cancerous sample, or betweensamples from different sources.

Alternatively, the expression level can be provided as a relativeexpression level. To determine a relative expression level of a marker,the level of expression of the marker is determined for 10 or moresamples of normal versus cancer cell isolates, preferably 50 or moresamples, prior to the determination of the expression level for thesample in question. The mean expression level of each of the genesassayed in the larger number of samples is determined and this is usedas a baseline expression level for the marker. The expression level ofthe marker determined for the test sample (absolute level of expression)is then divided by the mean expression value obtained for that marker.This provides a relative expression level.

Preferably, the samples used in the baseline determination will be fromcancer cells or normal cells of the same tissue type. The choice of thecell source is dependent on the use of the relative expression level.Using expression found in normal tissues as a mean expression score aidsin validating whether the marker assayed is specific to the tissue fromwhich the cell was derived (versus normal cells). In addition, as moredata is accumulated, the mean expression value can be revised, providingimproved relative expression values based on accumulated data.Expression data from normal cells provides a means for grading theseverity of the cancer state.

In another preferred embodiment, expression of a marker is assessed bypreparing genomic DNA or mRNA/cDNA (i.e. a transcribed polynucleotide)from cells in a subject sample, and by hybridizing the genomic DNA ormRNA/cDNA with a reference polynucleotide which is a complement of apolynucleotide comprising the marker, and fragments thereof. cDNA can,optionally, be amplified using any of a variety of polymerase chainreaction methods prior to hybridization with the referencepolynucleotide. Expression of one or more markers can likewise bedetected using quantitative PCR (QPCR) to assess the level of expressionof the marker(s). Alternatively, any of the many known methods ofdetecting mutations or variants (e.g. single nucleotide polymorphisms,deletions, etc.) of a marker of the invention may be used to detectoccurrence of a mutated marker in a subject.

In a related embodiment, a mixture of transcribed polynucleotidesobtained from the sample is contacted with a substrate having fixedthereto a polynucleotide complementary to or homologous with at least aportion (e.g. at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or morenucleotide residues) of a marker of the invention. If polynucleotidescomplementary to or homologous with are differentially detectable on thesubstrate (e.g. detectable using different chromophores or fluorophores,or fixed to different selected positions), then the levels of expressionof a plurality of markers can be assessed simultaneously using a singlesubstrate (e.g. a “gene chip” microarray of polynucleotides fixed atselected positions). When a method of assessing marker expression isused which involves hybridization of one nucleic acid with another, itis preferred that the hybridization be performed under stringenthybridization conditions.

In another embodiment, a combination of methods to assess the expressionof a marker is utilized.

Because the compositions, kits, and methods of the invention rely ondetection of a difference in expression levels or copy number of one ormore markers of the invention, it is preferable that the level ofexpression or copy number of the marker is significantly greater thanthe minimum detection limit of the method used to assess expression orcopy number in at least one of normal cells and cancerous cells.

3. Methods for Detection of Expressed Protein

The activity or level of a marker protein can also be detected and/orquantified by detecting or quantifying the expressed polypeptide. Thepolypeptide can be detected and quantified by any of a number of meanswell known to those of skill in the art. These may include analyticbiochemical methods such as electrophoresis, capillary electrophoresis,high performance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, and the like, or variousimmunological methods such as fluid or gel precipitin reactions,immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, western blotting, and the like. A skilledartisan can readily adapt known protein/antibody detection methods foruse in determining whether cells express a marker of the presentinvention.

A preferred agent for detecting a polypeptide of the invention is anantibody capable of binding to a polypeptide corresponding to a markerof the invention, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin.

In a preferred embodiment, the antibody is labeled, e.g. aradio-labeled, chromophore-labeled, fluorophore-labeled, orenzyme-labeled antibody). In another embodiment, an antibody derivative(e.g. an antibody conjugated with a substrate or with the protein orligand of a protein-ligand pair {e.g. biotin-streptavidin}), or anantibody fragment (e.g. a single-chain antibody, an isolated antibodyhypervariable domain, etc.) which binds specifically with a proteincorresponding to the marker, such as the protein encoded by the openreading frame corresponding to the marker or such a protein which hasundergone all or a portion of its normal post-translationalmodification, is used.

Proteins from cells can be isolated using techniques that are well knownto those of skill in the art. The protein isolation methods employedcan, for example, be such as those described in Harlow and Lane (Harlowand Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.).

In one format, antibodies, or antibody fragments, can be used in methodssuch as Western blots or immunofluorescence techniques to detect theexpressed proteins. In such uses, it is generally preferable toimmobilize either the antibody or proteins on a solid support. Suitablesolid phase supports or carriers include any support capable of bindingan antigen or an antibody. Well-known supports or carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite.

One skilled in the art will know many other suitable carriers forbinding antibody or antigen, and will be able to adapt such support foruse with the present invention. For example, protein isolated from cellscan be run on a polyacrylamide gel electrophoresis and immobilized ontoa solid phase support such as nitrocellulose. The support can then bewashed with suitable buffers followed by treatment with the detectablylabeled antibody. The solid phase support can then be washed with thebuffer a second time to remove unbound antibody. The amount of boundlabel on the solid support can then be detected by conventional means.Means of detecting proteins using electrophoretic techniques are wellknown to those of skill in the art (see generally, R. Scopes (1982)Protein Purification, Springer-Verlag, N.Y.; Deutscher, (1990) Methodsin Enzymology Vol. 182: Guide to Protein Purification, Academic Press,Inc., N.Y.).

In another preferred embodiment, Western blot (immunoblot) analysis isused to detect and quantify the presence of a polypeptide in the sample.This technique generally comprises separating sample proteins by gelelectrophoresis on the basis of molecular weight, transferring theseparated proteins to a suitable solid support, (such as anitrocellulose filter, a nylon filter, or derivatized nylon filter), andincubating the sample with the antibodies that specifically bind apolypeptide. The anti-polypeptide antibodies specifically bind to thepolypeptide on the solid support. These antibodies may be directlylabeled or alternatively may be subsequently detected using labeledantibodies (e.g., labeled sheep anti-mouse antibodies) that specificallybind to the anti-polypeptide.

In a more preferred embodiment, the polypeptide is detected using animmunoassay. As used herein, an immunoassay is an assay that utilizes anantibody to specifically bind to the analyte. The immunoassay is thuscharacterized by detection of specific binding of a polypeptide to ananti-antibody as opposed to the use of other physical or chemicalproperties to isolate, target, and quantify the analyte.

The polypeptide is detected and/or quantified using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Asai (1993) Methods in Cell BiologyVolume 37: Antibodies in Cell Biology, Academic Press, Inc. New York;Stites & Terr (1991) Basic and Clinical Immunology 7th Edition.

Immunological binding assays (or immunoassays) typically utilize a“capture agent” to specifically bind to and often immobilize the analyte(polypeptide or subsequence). The capture agent is a moiety thatspecifically binds to the analyte. In a preferred embodiment, thecapture agent is an antibody that specifically binds a polypeptide. Theantibody (anti-peptide) may be produced by any of a number of means wellknown to those of skill in the art.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Thus, the labeling agent may be a labeledpolypeptide or a labeled anti-antibody. Alternatively, the labelingagent may be a third moiety, such as another antibody, that specificallybinds to the antibody/polypeptide complex.

In one preferred embodiment, the labeling agent is a second humanantibody bearing a label. Alternatively, the second antibody may lack alabel, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second can be modified with a detectable moiety, e.g. asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, andAkerstrom (1985) J. Immunol., 135: 2589-2542).

As indicated above, immunoassays for the detection and/or quantificationof a polypeptide can take a wide variety of formats well known to thoseof skill in the art.

Preferred immunoassays for detecting a polypeptide are eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of captured analyte is directly measured. In onepreferred “sandwich” assay, for example, the capture agent (anti-peptideantibodies) can be bound directly to a solid substrate where they areimmobilized. These immobilized antibodies then capture polypeptidepresent in the test sample. The polypeptide thus immobilized is thenbound by a labeling agent, such as a second human antibody bearing alabel.

In competitive assays, the amount of analyte (polypeptide) present inthe sample is measured indirectly by measuring the amount of an added(exogenous) analyte (polypeptide) displaced (or competed away) from acapture agent (anti peptide antibody) by the analyte present in thesample. In one competitive assay, a known amount of, in this case, apolypeptide is added to the sample and the sample is then contacted witha capture agent. The amount of polypeptide bound to the antibody isinversely proportional to the concentration of polypeptide present inthe sample.

In one particularly preferred embodiment, the antibody is immobilized ona solid substrate. The amount of polypeptide bound to the antibody maybe determined either by measuring the amount of polypeptide present in apolypeptide/antibody complex, or alternatively by measuring the amountof remaining uncomplexed polypeptide. The amount of polypeptide may bedetected by providing a labeled polypeptide.

The assays of this invention are scored (as positive or negative orquantity of polypeptide) according to standard methods well known tothose of skill in the art. The particular method of scoring will dependon the assay format and choice of label. For example, a Western Blotassay can be scored by visualizing the colored product produced by theenzymatic label. A clearly visible colored band or spot at the correctmolecular weight is scored as a positive result, while the absence of aclearly visible spot or band is scored as a negative. The intensity ofthe band or spot can provide a quantitative measure of polypeptide.

Antibodies for use in the various immunoassays described herein, can beproduced as described below.

In another embodiment, level (activity) is assayed by measuring theenzymatic activity of the gene product. Methods of assaying the activityof an enzyme are well known to those of skill in the art.

In vivo techniques for detection of a biomarker protein includeintroducing into a subject a labeled antibody directed against theprotein. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

Certain markers identified by the methods of the invention may besecreted proteins. It is a simple matter for the skilled artisan todetermine whether any particular marker protein is a secreted protein.In order to make this determination, the marker protein is expressed in,for example, a mammalian cell, preferably a human cell line,extracellular fluid is collected, and the presence or absence of theprotein in the extracellular fluid is assessed (e.g. using a labeledantibody which binds specifically with the protein).

The following is an example of a method which can be used to detectsecretion of a protein. About 8×10⁵ 293T cells are incubated at 37° C.in wells containing growth medium (Dulbecco's modified Eagle's medium{DMEM} supplemented with 10% fetal bovine serum) under a 5% (v/v) CO2,95% air atmosphere to about 60-70% confluence. The cells are thentransfected using a standard transfection mixture comprising 2micrograms of DNA comprising an expression vector encoding the proteinand 10 microliters of LipofectAMINE™ (GIBCO/BRL Catalog no. 18342-012)per well. The transfection mixture is maintained for about 5 hours, andthen replaced with fresh growth medium and maintained in an airatmosphere. Each well is gently rinsed twice with DMEM which does notcontain methionine or cysteine (DMEM-MC; ICN Catalog no. 16-424-54).About 1 milliliter of DMEM-MC and about 50 microcuries of Trans-³⁵S™reagent (ICN Catalog no. 51006) are added to each well. The wells aremaintained under the 5% CO₂ atmosphere described above and incubated at37° C. for a selected period. Following incubation, 150 microliters ofconditioned medium is removed and centrifuged to remove floating cellsand debris. The presence of the protein in the supernatant is anindication that the protein is secreted.

It will be appreciated that subject samples, e.g., a sample containingtissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinalfluid, urine, stool, bile, pancreatic juice, and pancreatic tissue, maycontain cells therein, particularly when the cells are cancerous, and,more particularly, when the cancer is metastasizing, and thus may beused in the methods of the present invention. The cell sample can, ofcourse, be subjected to a variety of well-known post-collectionpreparative and storage techniques (e.g., nucleic acid and/or proteinextraction, fixation, storage, freezing, ultrafiltration, concentration,evaporation, centrifugation, etc.) prior to assessing the level ofexpression of the marker in the sample. Thus, the compositions, kits,and methods of the invention can be used to detect expression of markerscorresponding to proteins having at least one portion which is displayedon the surface of cells which express it. It is a simple matter for theskilled artisan to determine whether the protein corresponding to anyparticular marker comprises a cell-surface protein. For example,immunological methods may be used to detect such proteins on wholecells, or well known computer-based sequence analysis methods (e.g. theSIGNALP program; Nielsen et al., 1997, Protein Engineering 10:1-6) maybe used to predict the presence of at least one extracellular domain(i.e. including both secreted proteins and proteins having at least onecell-surface domain). Expression of a marker corresponding to a proteinhaving at least one portion which is displayed on the surface of a cellwhich expresses it may be detected without necessarily lysing the cell(e.g. using a labeled antibody which binds specifically with acell-surface domain of the protein).

The invention also encompasses kits for detecting the presence of apolypeptide or nucleic acid corresponding to a marker of the inventionin a biological sample, e.g., a sample containing tissue, whole blood,serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool,bile, pancreatic juice, and pancreatic tissue. Such kits can be used todetermine if a subject is suffering from or is at increased risk ofdeveloping cancer. For example, the kit can comprise a labeled compoundor agent capable of detecting a polypeptide or an mRNA encoding apolypeptide corresponding to a marker of the invention in a biologicalsample and means for determining the amount of the polypeptide or mRNAin the sample (e.g., an antibody which binds the polypeptide or anoligonucleotide probe which binds to DNA or mRNA encoding thepolypeptide). Kits can also include instructions for interpreting theresults obtained using the kit.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to apolypeptide corresponding to a marker of the invention; and, optionally,(2) a second, different antibody which binds to either the polypeptideor the first antibody and is conjugated to a detectable label.

For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptidecorresponding to a marker of the invention or (2) a pair of primersuseful for amplifying a nucleic acid molecule corresponding to a markerof the invention. The kit can also comprise, e.g., a buffering agent, apreservative, or a protein stabilizing agent. The kit can furthercomprise components necessary for detecting the detectable label (e.g.,an enzyme or a substrate). The kit can also contain a control sample ora series of control samples which can be assayed and compared to thetest sample. Each component of the kit can be enclosed within anindividual container and all of the various containers can be within asingle package, along with instructions for interpreting the results ofthe assays performed using the kit.

4. Method for Detecting Structural Alterations

The invention also provides a method for assessing whether a subject isafflicted with cancer or is at risk for developing cancer by comparingthe structural alterations, e.g., mutations or allelic variants, of amarker in a cancer sample with the structural alterations, e.g.,mutations of a marker in a normal, e.g., control sample. The presence ofa structural alteration, e.g., mutation or allelic variant in the markerin the cancer sample is an indication that the subject is afflicted withcancer.

A preferred detection method is allele specific hybridization usingprobes overlapping the polymorphic site and having about 5, 10, 20, 25,or 30 nucleotides around the polymorphic region. In a preferredembodiment of the invention, several probes capable of hybridizingspecifically to allelic variants are attached to a solid phase support,e.g., a “chip”. Oligonucleotides can be bound to a solid support by avariety of processes, including lithography. For example a chip can holdup to 250,000 oligonucleotides (GeneChip, Affymetrix™). Mutationdetection analysis using these chips comprising oligonucleotides, alsotermed “DNA probe arrays” is described e.g., in Cronin et al. (1996)Human Mutation 7:244. In one embodiment, a chip comprises all theallelic variants of at least one polymorphic region of a gene. The solidphase support is then contacted with a test nucleic acid andhybridization to the specific probes is detected. Accordingly, theidentity of numerous allelic variants of one or more genes can beidentified in a simple hybridization experiment. For example, theidentity of the allelic variant of the nucleotide polymorphism in the 5′upstream regulatory element can be determined in a single hybridizationexperiment.

In other detection methods, it is necessary to first amplify at least aportion of a marker prior to identifying the allelic variant.Amplification can be performed, e.g., by PCR and/or LCR (see Wu andWallace (1989) Genomics 4:560), according to methods known in the art.In one embodiment, genomic DNA of a cell is exposed to two PCR primersand amplification for a number of cycles sufficient to produce therequired amount of amplified DNA. In preferred embodiments, the primersare located between 150 and 350 base pairs apart.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al., (1988) Bio/Technology 6:1197), andself-sustained sequence replication (Guatelli et al., (1989)Proc. Nat.Acad. Sci. 87:1874), and nucleic acid based sequence amplification(NABSA), or any other nucleic acid amplification method, followed by thedetection of the amplified molecules using techniques well known tothose of skill in the art. These detection schemes are especially usefulfor the detection of nucleic acid molecules if such molecules arepresent in very low numbers.

In one embodiment, any of a variety of sequencing reactions known in theart can be used to directly sequence at least a portion of a marker anddetect allelic variants, e.g., mutations, by comparing the sequence ofthe sample sequence with the corresponding reference (control) sequence.Exemplary sequencing reactions include those based on techniquesdeveloped by Maxam and Gilbert (Proc. Natl. Acad Sci USA (1977) 74:560)or Sanger (Sanger et al. (1977) Proc. Nat. Acad. Sci. 74:5463). It isalso contemplated that any of a variety of automated sequencingprocedures may be utilized when performing the subject assays(Biotechniques (1995) 19:448), including sequencing by mass spectrometry(see, for example, U.S. Pat. No. 5,547,835 and international patentapplication Publication Number WO 94/16101, entitled DNA Sequencing byMass Spectrometry by H. Köster; U.S. Pat. No. 5,547,835 andinternational patent application Publication Number WO 94/21822 entitled“DNA Sequencing by Mass Spectrometry Via Exonuclease Degradation” by H.Köster), and U.S. Pat. No. 5,605,798 and International PatentApplication No. PCT/US96/03651 entitled DNA Diagnostics Based on MassSpectrometry by H. Köster; Cohen et al. (1996) Adv Chromatogr36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol38:147-159). It will be evident to one skilled in the art that, forcertain embodiments, the occurrence of only one, two or three of thenucleic acid bases need be determined in the sequencing reaction. Forinstance, A-track or the like, e.g., where only one nucleotide isdetected, can be carried out.

Yet other sequencing methods are disclosed, e.g., in U.S. Pat. No.5,580,732 entitled “Method of DNA sequencing employing a mixedDNA-polymer chain probe” and U.S. Pat. No. 5,571,676 entitled “Methodfor mismatch-directed in vitro DNA sequencing.”

In some cases, the presence of a specific allele of a marker in DNA froma subject can be shown by restriction enzyme analysis. For example, aspecific nucleotide polymorphism can result in a nucleotide sequencecomprising a restriction site which is absent from the nucleotidesequence of another allelic variant.

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetroxide and with piperidine) can beused to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, thetechnique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing a control nucleic acid, which is optionallylabeled, e.g., RNA or DNA, comprising a nucleotide sequence of a markerallelic variant with a sample nucleic acid, e.g., RNA or DNA, obtainedfrom a tissue sample. The double-stranded duplexes are treated with anagent which cleaves single-stranded regions of the duplex such asduplexes formed based on basepair mismatches between the control andsample strands. For instance, RNA/DNA duplexes can be treated with RNaseand DNA/DNA hybrids treated with S1 nuclease to enzymatically digest themismatched regions. In other embodiments, either DNA/DNA or RNA/DNAduplexes can be treated with hydroxylamine or osmium tetroxide and withpiperidine in order to digest mismatched regions. After digestion of themismatched regions, the resulting material is then separated by size ondenaturing polyacrylamide gels to determine whether the control andsample nucleic acids have an identical nucleotide sequence or in whichnucleotides they are different. See, for example, Cotton et al (1988)Proc. Natl Acad Sci USA 85:4397; Saleeba et al (1992) Methods Enzymol.217:286-295. In a preferred embodiment, the control or sample nucleicacid is labeled for detection.

In another embodiment, an allelic variant can be identified bydenaturing high-performance liquid chromatography (DHPLC) (Oefner andUnderhill, (1995) Am. J. Human Gen. 57:Suppl. A266). DHPLC usesreverse-phase ion-pairing chromatography to detect the heteroduplexesthat are generated during amplification of PCR fragments fromindividuals who are heterozygous at a particular nucleotide locus withinthat fragment (Oefner and Underhill (1995) Am. J. Human Gen. 57:Suppl.A266). In general, PCR products are produced using PCR primers flankingthe DNA of interest. DHPLC analysis is carried out and the resultingchromatograms are analyzed to identify base pair alterations ordeletions based on specific chromatographic profiles (see O'Donovan etal. (1998) Genomics 52:44-49).

In other embodiments, alterations in electrophoretic mobility is used toidentify the type of marker allelic variant. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci. USA 86:2766, see also Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control nucleicacids are denatured and allowed to renature. The secondary structure ofsingle-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In anotherpreferred embodiment, the subject method utilizes heteroduplex analysisto separate double stranded heteroduplex molecules on the basis ofchanges in electrophoretic mobility (Keen et al. (1991) Trends Genet.7:5).

In yet another embodiment, the identity of an allelic variant of apolymorphic region is obtained by analyzing the movement of a nucleicacid comprising the polymorphic region in polyacrylamide gels containinga gradient of denaturant is assayed using denaturing gradient gelelectrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGEis used as the method of analysis, DNA will be modified to insure thatit does not completely denature, for example by adding a GC clamp ofapproximately 40 bp of high-melting GC-rich DNA by PCR. In a furtherembodiment, a temperature gradient is used in place of a denaturingagent gradient to identify differences in the mobility of control andsample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:1275).

Examples of techniques for detecting differences of at least onenucleotide between two nucleic acids include, but are not limited to,selective oligonucleotide hybridization, selective amplification, orselective primer extension. For example, oligonucleotide probes may beprepared in which the known polymorphic nucleotide is placed centrally(allele-specific probes) and then hybridized to target DNA underconditions which permit hybridization only if a perfect match is found(Saiki et al. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl.Acad. Sci. USA 86:6230; and Wallace et al. (1979) Nucl. Acids Res.6:3543). Such allele specific oligonucleotide hybridization techniquesmay be used for the simultaneous detection of several nucleotide changesin different polylmorphic regions of marker. For example,oligonucleotides having nucleotide sequences of specific allelicvariants are attached to a hybridizing membrane and this membrane isthen hybridized with labeled sample nucleic acid. Analysis of thehybridization signal will then reveal the identity of the nucleotides ofthe sample nucleic acid.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the allelic variant of interest in the center of the molecule(so that amplification depends on differential hybridization) (Gibbs etal (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end ofone primer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238; Newton etal. (1989) Nucl. Acids Res. 17:2503). This technique is also termed“PROBE” for Probe Oligo Base Extension. In addition it may be desirableto introduce a novel restriction site in the region of the mutation tocreate cleavage-based detection (Gasparini et al (1992) Mol. Cell.Probes 6:1).

In another embodiment, identification of the allelic variant is carriedout using an oligonucleotide ligation assay (OLA), as described, e.g.,in U.S. Pat. No. 4,998,617 and in Landegren, U. et al., (1988) Science241:1077-1080. The OLA protocol uses two oligonucleotides which aredesigned to be capable of hybridizing to abutting sequences of a singlestrand of a target. One of the oligonucleotides is linked to aseparation marker, e.g., biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al., (1990)Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927. In this method, PCR isused to achieve the exponential amplification of target DNA, which isthen detected using OLA.

The invention further provides methods for detecting single nucleotidepolymorphisms in a marker. Because single nucleotide polymorphismsconstitute sites of variation flanked by regions of invariant sequence,their analysis requires no more than the determination of the identityof the single nucleotide present at the site of variation and it isunnecessary to determine a complete gene sequence for each subject.Several methods have been developed to facilitate the analysis of suchsingle nucleotide polymorphisms.

In one embodiment, the single base polymorphism can be detected by usinga specialized exonuclease-resistant nucleotide, as disclosed, e.g., inMundy, C. R. (U.S. Pat. No. 4,656,127). According to the method, aprimer complementary to the allelic sequence immediately 3′ to thepolymorphic site is permitted to hybridize to a target molecule obtainedfrom a particular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of a polymorphic site.Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087).As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employedthat is complementary to allelic sequences immediately 3′ to apolymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

Several primer-guided nucleotide incorporation procedures for assayingpolymorphic sites in DNA have been described (Komher, J. S. et al.,(1989) Nucl. Acids. Res. 17:7779-7784; Sokolov, B. P., (1990) Nucl.Acids Res. 18:3671; Syvanen, A.-C., et al., (990) Genomics 8:684-692;Kuppuswamy, M. N. et al., (1991) Proc. Natl. Acad. Sci. (U.S.A.)88:1143-1147; Prezant, T. R. et al., (1992) Hum. Mutat. 1:159-164;Ugozzoli, L. et al., (1992) GATA 9:107-112; Nyren, P. (1993) et al.,Anal Biochem. 208:171-175). These methods differ from GBA™ in that theyall rely on the incorporation of labeled deoxynucleotides todiscriminate between bases at a polymorphic site. In such a format,since the signal is proportional to the number of deoxynucleotidesincorporated, polymorphisms that occur in runs of the same nucleotidecan result in signals that are proportional to the length of the run(Syvanen, A. C., et al., (1993) Amer. J. Hum. Genet. 52:46-59).

For determining the identity of the allelic variant of a polymorphicregion located in the coding region of a marker, yet other methods thanthose described above can be used. For example, identification of anallelic variant which encodes a mutated marker can be performed by usingan antibody specifically recognizing the mutant protein in, e.g.,immunohistochemistry or immunoprecipitation. Antibodies to wild-typemarker or mutated forms of markers can be prepared according to methodsknown in the art.

Alternatively, one can also measure an activity of a marker, such asbinding to a marker ligand. Binding assays are known in the art andinvolve, e.g., obtaining cells from a subject, and performing bindingexperiments with a labeled ligand, to determine whether binding to themutated form of the protein differs from binding to the wild-type of theprotein.

B. Pharmacogenomics

Agents or modulators which have a stimulatory or inhibitory effect onamount and/or activity of a marker of the invention can be administeredto individuals to treat (prophylactically or therapeutically) cancer inthe subject. In conjunction with such treatment, the pharmacogenomics(i.e., the study of the relationship between an individual's genotypeand that individual's response to a foreign compound or drug) of theindividual may be considered. Differences in metabolism of therapeuticscan lead to severe toxicity or therapeutic failure by altering therelation between dose and blood concentration of the pharmacologicallyactive drug. Thus, the pharmacogenomics of the individual permits theselection of effective agents (e.g., drugs) for prophylactic ortherapeutic treatments based on a consideration of the individual'sgenotype. Such pharmacogenomics can further be used to determineappropriate dosages and therapeutic regimens. Accordingly, the amount,structure, and/or activity of the invention in an individual can bedetermined to thereby select appropriate agent(s) for therapeutic orprophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant variations in theresponse to drugs due to altered drug disposition and abnormal action inaffected persons. See, e.g., Linder (1997) Clin. Chem. 43(2):254-266. Ingeneral, two types of pharmacogenetic conditions can be differentiated.Genetic conditions transmitted as a single factor altering the way drugsact on the body are referred to as “altered drug action.” Geneticconditions transmitted as single factors altering the way the body actson drugs are referred to as “altered drug metabolism”. Thesepharmacogenetic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD)deficiency is a common inherited enzymopathy in which the main clinicalcomplication is hemolysis after ingestion of oxidant drugs(anti-malarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some subjectsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the amount, structure, and/or activity of a marker of theinvention in an individual can be determined to thereby selectappropriate agent(s) for therapeutic or prophylactic treatment of theindividual. In addition, pharmacogenetic studies can be used to applygenotyping of polymorphic alleles encoding drug-metabolizing enzymes tothe identification of an individual's drug responsiveness phenotype.This knowledge, when applied to dosing or drug selection, can avoidadverse reactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a modulator ofamount, structure, and/or activity of a marker of the invention.

C. Monitoring Clinical Trials

Monitoring the influence of agents (e.g., drug compounds) on amount,structure, and/or activity of a marker of the invention can be appliednot only in basic drug screening, but also in clinical trials. Forexample, the effectiveness of an agent to affect marker amount,structure, and/or activity can be monitored in clinical trials ofsubjects receiving treatment for cancer. In a preferred embodiment, thepresent invention provides a method for monitoring the effectiveness oftreatment of a subject with an agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, antibody, nucleic acid, antisensenucleic acid, ribozyme, small molecule, RNA interfering agent, or otherdrug candidate) comprising the steps of (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the amount, structure, and/or activity of one ormore selected markers of the invention in the pre-administration sample;(iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the amount, structure, and/or activity of themarker(s) in the post-administration samples; (v) comparing the level ofexpression of the marker(s) in the pre-administration sample with theamount, structure, and/or activity of the marker(s) in thepost-administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent can be desirable to increaseamount and/or activity of the marker(s) to higher levels than detected,i.e., to increase the effectiveness of the agent. Alternatively,decreased administration of the agent can be desirable to decreaseamount and/or activity of the marker(s) to lower levels than detected,i.e., to decrease the effectiveness of the agent.

EXAMPLES

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,figures, Sequence Listing, patents and published patent applicationscited throughout this application are hereby incorporated by reference.

Example 1

A. Materials and Methods

Primary Tumors and Cell Lines

All cell lines were acquired from the American Type Culture Collection(ATCC) or the German Collection of Microorganisms and Cell Cultures(DSMZ). All fresh-frozen specimens of primary pancreatic ductaladenocarcinoma were obtained from the Memorial Sloan-Kettering CancerCenter tumor bank and histology was confirmed by hematoxylin and eosin(H&E) staining prior to inclusion in the study (Table 2). Table 2 liststhe cell lines analyzed by array-CGH and expression profiling in thestudy.

Array-CGH Profiling on cDNA Microarrays

Genomic DNA was fragmented and random-prime labeled according topublished protocol (Pollack, J. R., et al. (1999) Nat Genet. 23, 41-6)with modifications. Labeled DNAs were hybridized to human cDNAmicroarrays containing 14,160 cDNA clones (Agilent Technologies™, Human1 clone set), for which approximately 9,420 unique map positions weredefined (NCBI, Build 33). The median interval between mapped elements is100.1 kilobase, 92.8% of intervals are less than 1 megabase, and 98.6%are less than 3 megabases.

Fluorescence ratios of scanned images of the arrays were calculated andthe raw array-CGH profiles were processed to identify statisticallysignificant transitions in copy number using a segmentation algorithmwhich employs permutation to determine the significance of change pointsin the raw data (Olshen, A. B., and Venkatraman, E. S. (2002) ASAProceedings of the Joint Statistical Meetings, 2530-2535; Ginzinger, D.G. (2002) Exp Hematol 30, 503-12). Each segment was assigned a Log₂ratio that is the median of the contained probes. The data was centeredby the tallest mode in the distribution of the segmented values. Aftermode-centering, gains and losses were defined as Log₂ ratios of greaterthan or equal to +0.13 or −0.13 (+/−4 standard deviations of the middle50% quantile of data), and amplification and deletion as ratio greaterthan 0.52 or less than −0.58, respectively (i.e., 97% or 3% quantiles)(FIG. 4).

Automated Locus Definition

Loci were defined by an automated algorithm applied to the segmenteddata based on the following rules:

1. Segments above 97^(th) percentile or below 3^(rd) percentile wereidentified as altered.

2. If two or more altered segments are adjacent in a single profile orseparated by less than 500 KB, the entire region spanned by the segmentswas considered to be an altered span.

3. Highly altered segments or spans that were shorter than 20 MB wereretained as “informative spans” for defining discrete locus boundaries.Longer regions were not discarded, but were not included in defininglocus boundaries.

4. Informative spans were compared across samples to identifyoverlapping groups of positive-value or negative-value segments; eachgroup defined a locus.

5. Minimal common regions (MCRs) were defined as contiguous spans havingat least 75% of the peak recurrence as calculated by counting theoccurrence of highly altered segments. If two MCRs were separated by agap of only one probe position, they were joined. If there were morethan 3 MCRs in a locus, the whole region was reported as a singlecomplex MCR.

MCR Characterization

For each MCR, the peak segment value was identified. Recurrence of gainor loss was calculated across all samples based on the lower thresholdspreviously defined (˜+/−0.13). As an additional measure of recurrenceindependent of thresholds for segment value or length, Median Aberration(MA) was calculated for each probe position by taking the median of allsegment values above zero for amplified regions, below zero for deletedregions. This pair of values was compared to the distribution of valuesobtained after permuting the probe labels independently in each sampleprofile. Where the magnitude of the MA exceeds 95% of the permutedaverages, the region was marked as significantly gained or lost, andthis was used in the voting system for prioritization. The number ofknown genes and GENSCAN model predicted genes were counted based on theApril 2003 human assembly at UCSC (genome.ucsc.edu).

QPCR Verification

PCR primers were designed to amplify products of 100-150 bp withintarget and control sequences. Primers for control sequences in each cellline were designed within a region of euploid copy number as shown byarray-CGH analysis. Quantitative PCR was performed by monitoring inreal-time the increase in fluorescence of SYBR Green dye (Qiagen™) withan ABI 7700 sequence detection system (Perkin Elmer Life Sciences™).Relative gene copy number was calculated by the comparative C_(t) method(Ginzinger, D. G. (2002) Exp Hematol 30, 503-12).

Expression Profiling on Affymetrix GeneChip™

Biotinylated target cRNA was generated from total sample RNA andhybridized to human oligonucleotide probe arrays (U133A, Affymetrix™,Santa Clara, Calif.) according to standard protocols (Golub, T. R., etal. (1999) Science 286, 531-7). Expression values for each gene werestandardized by Log2 ratio to a middle value for the sample set, definedas the midpoint between 25% and 75% quantiles, and were mapped togenomic positions based on NCBI Build 33 of the human genome.

Integrated Copy Number and Expression Analysis

Array-CGH data was interpolated such that each expression value can bemapped to its corresponding copy number value. In order to maximizedetection of focal CNAs, two separate interpolations were calculated:one selecting the higher bounding CGH probe and one choosing the lower.For each gene position, the samples were grouped based on whetherarray-CGH shows altered copy number or not based on interpolated CGHvalue. The effect of gene dosage on expression was measured bycalculating a gene weight defined as the difference of the means of theexpression value in the altered and unaltered sample groups divided bythe sum of the standard deviations of the expression values in alteredand unaltered sample groups (Hyman, E., et al. (2002) Cancer Res 62,6240-5). The significance of the weight for each gene was estimated bypermuting the sample labels 10,000 times and applying an alpha thresholdof 0.05.

B. Results

Comprehensive Catalogue of CNAs in the Pancreatic Adenocarcinoma Genome

From a total of 75 primary pancreatic tumor specimens, 13 samples wereidentified that possessed greater than 60% neoplastic cellularity.Genomic DNAs from these primary tumor samples, along with DNAs derivedfrom 24 established pancreatic cancer cell lines, e.g., pancreaticadenocarcinoma cell lines (Table 2), were subjected to genome-widearray-CGH profiling using a cDNA-based array platform that offers amedian resolution of 100 kB. To facilitate identification of significantcopy number events in these array-CGH profiles, a modified version ofthe circular binary segmentation methodology developed by Olshen andcolleagues was employed (Olshen, A. B., and Venkatraman, E. S. (2002)ASA Proceedings of the Joint Statistical Meetings 2530-2535; Ginzinger,D. G. (2002) Exp Hematol 30, 503-12; Golub, T. R., et al. (1999) Science286, 531-7; Hyman, E., et al. (2002) Cancer Res 62, 6240-5; Lucito, R.,et al. (2003) Genome Res 13, 2291-305). This algorithm appliesnonparametric statistical testing to identify and distinguish discretecopy number transition points from chance noise events in the primarydataset. As shown in FIG. 1A, the segmented array-CGH profiles readilyidentified large regional changes that are typically of low amplitude,hereafter referred to as ‘gain’ or ‘loss’. Similarly, focal highamplitude alterations representing ‘amplification’ or ‘deletion’ areevident in both primary tumor specimens and tumor cell lines (FIG. 1).Recurrence frequencies of the CNAs reported here match the frequenciesdescribed in the published literature (Solinas-Toldo, S., et al. (1996)Cancer Res 56, 3803-7; Mahlamaki, E. H., et al. (1997) Genes ChromosomesCancer 20, 383-91; Mahlamaki, E. H., et al. (2002) Genes ChromosomesCancer 35, 353-8; Fukushige, S., et al. (1997) Genes Chromosomes Cancer19:161-9; Curtis, L. J., et al. (1998) Genomics 53, 42-55; Ghadimi, B.M., et al. (1999) Am J Pathol 154, 525-36; Armengol, G., et al. (2000)Cancer Genet Cytogenet 116, 133-41) (FIG. 1B). There is also strongconcordance between primary tumors and cell lines with respect to gainson 3q, 8q and 20q and losses on I p, 3p, 6q, 9p, 17p and 18q (see FIG. 5and FIG. 6). However, some differences were evident between primarytumor and cell line datasets and are likely attributable to the cellularheterogeneity within primary tumor samples and/or culture-inducedgenetic adaptation in the cell lines.

The identification of many novel CNAs, along with the high degree ofstructural complexity within each CNA, prompted the implementation ofobjective criteria to define and prioritize CNAs across the dataset. Tothat end, a locus-identification algorithm was developed that definesinformative CNAs on the basis of size and achievement of a highsignificance threshold for the amplitude of change. Overlapping CNAsfrom multiple profiles are then merged in an automated fashion to definea discrete “locus” of regional copy number change, the bounds of whichrepresent the combined physical extent of these overlapping CNAs (FIG.1C). Each locus is characterized by a peak profile, the width andamplitude of which reflect the contour of the most prominentamplification or deletion for that locus. Furthermore, within eachlocus, one or more minimal common region (MCRs) can be identified acrossmultiple tumor samples (FIG. 1C), with each MCR potentially harboring adistinct cancer-relevant gene targeted for copy number alteration acrossthe sample set.

The locus identification algorithm is highly effective in delineatingmore discrete CNAs within previously described larger regions of gain orloss. For example, chromosome 6q has been reported as one of the mostfrequently deleted regions in pancreatic adenocarcinoma, but novalidated tumor suppressor gene has yet been assigned to this locus.Analysis of 6q loss in the dataset presented herein has identified 5distinct MCRs that range in size from 2.4 to 12.8 Mb, raising thepossibility that there may be multiple targets for 6q loss. Notably, twoof these MCRs (Table 3, Locus #75 and #76) coincide with previouslyidentified regions of common allelic loss (Abe, T., et al. (1999) GenesChromosomes Cancer 25, 60-4), an observation that provides furthervalidation for the analytical approach described herein.

Selection of High-Priority Loci

The above locus-identification algorithm defined 287 discrete MCRs (from256 independent autosomal loci) within this dataset and annotated eachin terms of recurrence, amplitude of change and representation in bothcell lines and primary tumors. Based upon extensive experience with thisplatform across many tumor types, recurrence across multiple independentsamples and high amplitude signals are the two features most predictiveof verification by independent assays. Hence, these discrete MCRs wereprioritized based on four criteria that include (1) recurrence ofhigh-threshold amplification or deletion (above 97^(th) percentile orbelow 3^(rd) percentile) in at least two specimens, (2) presence of ahigh-threshold event in at least one primary tumor specimen, (3)statistically significant Median Aberration (see M&M), and (4) a peakamplitude of equal to or greater than absolute Log₂ value of 0.8 ineither a cell line or primary tumor (beyond 0.5% quantiles).

Implementation of this prioritization scheme yielded 64 MCRs within 54independent loci that satisfied at least three of the four criteria(Table 1). In Table 1, the high-confidence recurrent CNAs in pancreaticadenocarcinoma are depicted. For each MCR, the numbers of known (“K”)and predicted (“P”) (GenScan) transcripts (“Trspt”) are indicated. Ofthese, some are represented on Affymetrix™ U133A chip (“Total”), asubset of which show statistical significance (p<0.05) for copy numbercorrelation (“Sig”). MCR recurrence is denoted as percentage of thetotal dataset. The numbers of primary tumors (T) or cell lines (C) withgain or loss, and amplification or deletion, are listed, respectively.Candidate genes within a locus for which there is literature support forinvolvement in pancreatic cancer are listed. Black diamonds denote theloci where the peak did not fall within a defined MCR. MCRs in bold havebeen validated by QPCR. Notably, genes known to play important roles inthe pathogenesis of pancreatic adenocarcinoma—the p16^(INK4A) and TP53tumor suppressors and the MYC, KRAS2 and AKT2 oncogenes—were presentwithin these high-confidence loci (Table 1). Within the prioritizedMCRs, there was an average recurrence rate for gain/loss of 38% acrossthe entire dataset and the maxima or minima absolute Log₂ values for 34of these 64 MCRs are greater than 1.0, placing them significantly abovethe threshold defined for amplification or deletion (FIG. 4). In themajority of cases, the peak profile of a locus coincided with one of theMCRs (47 of 54 Loci, Table 1) (Albertson, D. G., et al. (2000) NatGenet. 25, 144-6). The median size of these 64 prioritized MCRs is 2.7Mb, with 21 MCRs (33%) spanning 1 Mb or less (Table 1). Residing withinthese 21 highly focal MCRs with a median size of 0.33 Mb, there are onaverage 15 annotated and 8 GENSCAN predicted genes, rendering themhighly attractive for target identification.

The confidence-level ascribed to these prioritized loci was furthervalidated by real-time quantitative PCR (QPCR), which demonstrated 100%concordance with 16 selected MCRs defined by array-CGH (Table 1). Forexample, the MCR of an amplified locus at 7q21.11-7q32.2 was readilyconfirmed by QPCR (FIG. 2A). Furthermore, QPCR analyses also verifiedthe structural details of complex CNAs reported by array-CGH. As shownin FIG. 2B, QPCR precisely mirrored each component of the complex 9p21locus in HUP-T3, including homozygous deletion of p16^(INK4A), the knowntarget for this CNA. Such detailed structural information will proveuseful in dissecting the mechanisms responsible for the genesis of thesecancer-associated chromosomal aberrations.

When high-priority MCRs in Table 1 were combined with an additional 81moderate-priority MCRs (within 66 distinct loci) satisfying 2 out of 4criteria, a genomic characterization produced a list of 145 MCRs within121 independent loci (Table 3). Table 3 shows the combined list of 145MCRs in 121 independent loci, including 64 high-confidence MCRs (≧3votes) and 81 moderate-priority (2 votes) MCRs, that were prioritized bythe automated algorithm as described herein. Each locus is assigned to acytogenetic band, while the actual extent of the locus is defined moreprecisely by its physical location on the chromosome (in Mb) (NCBI,Built 33). The overall contour of the locus is reflected by themaxima/minima profile, which defines the most prominent point ofamplification or deletion within the locus by its width (defined inphysical Mb) and amplitude. Each locus is furthered defined by one ormore Minimal Common Regions (MCRs), depending on the cytogeneticcomplexity of the locus. Each MCR is defined in a similar manner. Inaddition, the number of known and predicted (GENSCAN) transcripts aswell as the total number of Affymetrix-represented genes and those withp-value<0.05 are shown for each MCR. MCR recurrence is denoted aspercentage of the total dataset. The number of primary tumors (T) orcell lines (C) with gain or loss (90% and 10% quantiles, respectively)is listed. Furthermore, the subset of these tumors with gain/loss thatmeet the threshold for amplification or deletion (97% and 3% quantiles,respectively) are also indicated. The boundaries of the MCRs of eachlocus have been defined based on conservative parameters.

Integrated Analysis of Copy Number and Expression Information.

Copy number aberrations and their associated impact on gene expressionpatterns represent a common mechanism of oncogene activation and tumorsuppressor inactivation. Indeed, integration of copy number andtranscriptional profile datasets revealed a consistent influence of genedosage on mRNA expression globally across the genome (FIG. 6) (Hyman,E., et al. (2002) Cancer Res 62, 6240-5; Pollack, J. R., et al. (2002)Proc Natl Acad Sci USA 99, 12963-8). Conversely, as previouslydemonstrated (Platzer, P., et al. (2002) Cancer Res 62, 1134-8), only asubset of genes within any given CNA show copy-number-driven expressionchanges—a feature that provides a first-pass means of distinguishingbystanders from potential cancer gene targets within the CNA. As a casein point, a novel locus of amplification on chromosome 17 in the cellline Hup-T3 (Locus # 21, Table 1) contains 455 genes of which 151 arepresent on the Affymetrix U133A array. Of these 151 genes, only 19exhibited increased transcript levels above 2-fold. Moreover, these 19genes reside within the peak of this locus (FIG. 3A). Similarcorrelations can be established in regions of deletion. For example, the9p21 deletion locus in the BxPC-3 cell line demonstrated that only 5 outof 91 genes residing within the MCR show undetectable or decreasedexpression below 2-fold (FIG. 3B). Examination of p16^(INK4A), the knowntarget for deletion, across the entire sample set shows that 11 of 24cell lines show low or absent expression, of which 5 showed homozygousdeletion while the remaining 6 were present at the DNA level (FIG. 3C).In the latter, epigenetic silencing is the presumed mechanism ofp16^(INK4a) inactivation.

In many cancer types, including pancreatic adenocarcinoma, the truecell-of-origin remains unknown and thus a premalignant physiologicalframe-of-reference is not available. In the examples above, a model forinterfacing copy number and expression profiles by midpoint-centeringthe expression data and calculating a weighted statistic for assignmentof significance values to genes with correlated copy number andexpression was applied (Golub, T. R., et al. (1999) Science 286, 531-7;Hyman, E., et al. (2002) Cancer Res 62, 6240-5) (see M&M). Using thisapproach, the genes residing within the 64 high-confidence MCRs(Table 1) were prioritized based on the correlation of their expressionwith gene dosage. While only a subset of genes are represented, theAffymetrix™ U133A array permitted inclusion of 1,926 genes out of atotal of 4,742 genes residing within these MCRs for this analysis. Byweighing each of these 1,926 genes based on the magnitude of itsexpression alteration and its representation within CNAs across thedataset, the integrated copy number and expression analysis yielded alist of 603 genes that show a statistically significant associationbetween gene copy number and mRNA expression (p<0.05, Tables 4 and 5).Tables 4 and 5 show the genes located within high-priority MCRs(Table 1) and having highly significant correlation between geneexpression and gene dosage (p<0.05). The chromosome, physical positionin Mb, Gene Weight (see M&M) and p-value are listed. Affymetrix probe(s)number(s) corresponding to each GenBank Accession Number (“GI” Number)and UniGene ID are listed, along with SEQ ID NOs for each nucleic acidand protein sequence. P-Values were calculated by permutation analysisof a gene weight statistic. Of these, 336 are located within regions ofamplifications and 267 within regions of deletions. Importantly, amongthese 603 genes were known pancreatic cancer genes such as MYC (13),p16^(INK4A) (Rozenblum, E., et al. (1997) Cancer Res 57, 1731-4; Caldas,C., et al. (1994) Nat Genet. 8, 27-32) and DUSP6 (Furukawa, T., et al.(2003) Am J Pathol 162, 1807-15) (Tables 4 and 5), thus reinforcing thevalue of integrating both copy number and expression information.

While incomplete representation of known and predicted genes on theAffymetrix U133A expression array precluded assessment of all possibletarget genes, the complementary analysis of array-CGH and expressionprofiles presented herein serves to prioritize the list of availablecancer gene candidates and provides a basis for focus on a subset ofhigh-probability candidates. In addition, integrating genomic datasetsacross species may also prove effective in facilitating cancer geneidentification. A particularly productive path for oncogeneidentification may be the analysis of common integration sites (CISs)present in retrovirally-promoted leukemias and lymphomas (Neil, J. C. &Cameron, E. R. (2002) Cancer Cell 2, 253-5). Consistent with theparadigm that proviral integration primarily serves to activateendogenous proto-oncogene (Neil, J. C. & Cameron, E. R. (2002) CancerCell 2, 253-5), syntenic mapping of 232 CISs to the human genome (AkagiK., et al. (2004) Nucl. Acids Res 32) uncovered 19 CISs residing withinMCRs of amplified loci in Table 1, whereas only 10 would be expected bychance alone (p<0.006). On the contrary, MCRs within regions of loss ordeletion contained only 16 CIS's whereas 14.4 would have been expectedby chance alone. Thus, the abundance of CIS's mapping to amplified locimay represent genes with pathogenetic relevance in mouse models of tumorprogression as well as in human cancer, although there may be possiblecell-type specific roles for these candidate genes.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

TABLE 1 List of High-Confidence MCRs. Minimal Common Cytogenetic LocusLocus Peak Profile Regions (MCRs) # Band Boundary (Mb) Pos (MB) Max/MinPosition (MB) Size (MB) Gain and Amplification 1 1p13.1-p12116.83-119.49 119.07-119.2  1.56 116.83-119.49 2.65 2 5p15.33-p15.310.28-6.69 0.28-0.51 0.79 0.28-6.69 6.4 3 5q31.1-q31.1 133.51-134.33133.58-133.95 0.97 133.53-133.56 0.04 4 6p22.1-p21.32 28.12-32.7231.98-32.44 1.36 31.98-32.11 0.13 5 6p21.1-p21.1 42.91-43.19 42.98-43.150.84 42.91-43.03 0.12 6 7p22.3-p22.1 0.72-4.53 0.72-2.48 0.93 0.72-2.281.56 7 7p15.1-p14.3 30.12-31.56  30.5-30.81 0.82 30.12-31.66 1.44 87q11.21-q21.11 64.84-77.18 64.95-64.95 1.06 64.95-65.85 0.9 97q21.11-q32.2  79.45-129.46 97.86-98.55 3.06  92.33-112.27 19.94 108p12-p11.21  37.7-41.76 37.72-38.02 1.71  37.7-38.45 0.75 38.68-39.520.84 11 8q12.1-q12.3 59.09-63.68 59.23-62.26 0.58 59.23-60.02 1.6 128q21.3-q24.3  90.7-145.83 133.72-134.16 1.78 118.97-145.83 26.86 139p21.3-p13.2 23.68-37.87 35.65-36.56 0.86  35.6-36.56 0.96 1411q14.1-q14.2 78.15-86.74 82.76-85.89 0.9 83.39-86.21 2.82 1512p12.3-q13.13  16.6-53.06  20.7-22.54 2.52 21.82-22.39 0.57 1612q15-q15 68.27-68.87 68.44-68.77 1.38 68.27-68.85 0.59 1713q12.11-q14.13 18.68-43.95 19.21-24.22 0.89 20.65-24.85 4.19 1813q34-q34 112.84-113.06 112.85-113.01 0.82 112.84-113.06 0.23 1914q11.2-q24.3 18.82-74.47  73.56-74.47* 1.08 28.12-33.17 5.0552.41-63.53 11.12 20 17q12-q23.2 37.48-56.39 43.53-43.53 2.3738.95-55.26 16.31 21 17q23.2-q25.3 59.83-79.64  62.33-62.47* 3.6174.02-74.22 0.2 22 18p11.21-q12.1 12.02-28.55 19.67-21.37 2.1219.12-21.37 2.26 23 19p13.11-q13.32 23.32-50    45.03-45.39* 3.8441.41-44.6  3.2 24 19q13.32-q13.43 50.06-63.76 50.65-50.65 1.5950.06-62.89 12.83 25 20p13-q13.33  0.33-63.41 58.54-63.41 1.3425.68-31.48 5.8 58.24-63.41 6.17 26 22q11.1-q12.1 14.65-26.58 22.7-22.71.04 22.64-22.83 0.2 27 22q12.2-q12.2 28.75-29.85 28.96-29.83 0.9329.35-29.85 0.51 Loss and Deletion 28 1p36.21-p36.11 15.02-26.9521.46-21.46 −0.94 21.08-21.56 0.48 22.82-26.95 4.13 29 1p36.3-p34.328.37-39.18 32.79-33.04 −0.87 28.37-31.18 2.81 32.67-34.68 2.01 301p21.2-p21.1 100.63-103.3  103.25-103.25 −1.69 103.25-103.3  0.05 312p25.3-p24.3  0.21-16.09 10.92-11.3  −0.85  11.3-11.32 0.02 322p12-p11.2 75.23-89    79.31-85.51* −1.03  77.7-79.27 1.57 333p26.3-p24.3 1.33-18.8 13.85-14.52 −1 13.67-14.52 0.85 34 4q31.22-q32.1148.03-158.75 149.46-153.1  −0.93 151.71-154.37 2.65 155.09-158.56 3.4735 4q34.1-q35.2 174.84-188.09 175.03-187.79 −1.28 174.84-188.09 13.26 365q23.2-q23.3 127.55-130.53 127.65-128.48 −0.62 127.55-130.53 2.98 376q21-q22.31 106.63-119.43 107.02-116.57 −1.11 106.63-119.43 12.8 386q23.3-q24.3 135.17-146.81 137.46-138.13 −0.91 135.17-146.81 11.64 396q27-q27 168.07-170.64 168.63-170.64 −0.72 168.07-170.64 2.47 408p23.3-p12 2.06-37.7 2.06-2.1* −1.86 18.07-21.76 3.68 28.45-37.7  9.2541 9p24.3-p21.2  0.47-27.18 20.77-21.31 −2.63 0.47-3.39 2.92  6.3-23.6818.39 42 11q14.2-q14.3 85.89-89.24 86.21-89.24 −0.9 85.89-89.24 3.35 4312q12-q13.12 40.06-49.24 41.04-49.21 −0.71 40.06-49.24 9.18 4412q13.12-q13.3 49.65-55.82 50.01-53.06 −0.7 49.65-63.06 3.41 63.17-55.822.65 45 12q14.1-q15 67.92-68.77 62.76-68.27 −0.8 62.76-68.77 6.01 4612q21.2-q24.33 77.19-133.4 81.03-85.63 −1.19 77.19-91.44 14.25 4716p13.3-p12.2  0.03-23.87 0.76-23.7 −0.67 2.24-2.82 0.58 4817p13.3-q11.1  0.02-25.61  8.16-14.05 −0.99 10.36-12.8  2.44 4918q11.2-q21.1 18.61-46.28 34.95-40.68 −1.19 34.16-43.14 8.99 5018q22.1-q23  60.4-77.63 74.45-76.84 −1.53  60.4-77.63 17.23 5119q13.2-q13.43 44.67-63.76 69.99-60.04 −1.31 59.85-60.18 0.33 5221p11.2-q11.2  9.96-13.43  9.96-10.08 −1.24  9.96-13.43 3.47 5321q22.2-q22.3 39.76-46.94  41.7-41.76* −4.09 45.08-45.17 0.0946.77-46.94 0.17 54 22q11.1-q13.2 14.49-39.46 22.7-22.7 −1.3220.64-39.46 18.83 Minimal Common Regions (MCRs) MCR Recurrence # Trspt #on U133A Gain/Loss Amp/Del # Max Min K P Total Sig. % T C T C CandidatesGain and Amplification 1 1.55 23 47 15 2 23 2 6 2 2 2 1.05 57 164 19 843 1 14 0 5 3 0.97 0 0 1 0 14 1 4 1 1 4 1.36 15 3 14 1 29 1 9 0 4 NOTCH45 0.84 16 6 7 2 23 2 6 1 1 6 0.93 45 53 10 6 51 4 14 0 7 7 0.82 23 25 131 37 1 12 1 2 8 1.06 12 22 7 5 34 1 11 1 1 9 3.06 356 282 186 42 46 4 121 5 10 1.71 31 14 14 8 37 1 12 0 2 FGFRI 1.44 3 4 5 0 31 1 10 0 2 110.58 1 15 4 0 49 4 13 1 1 12 1.78 317 461 115 41 66 6 17 0 7 MYC 13 0.8639 23 18 9 20 1 6 0 3 14 0.9 18 26 9 0 46 0 16 0 2 15 2.52 9 5 3 1 26 27 2 2 KRAS2 16 1.38 15 13 1 0 23 2 6 1 2 17 0.89 43 74 19 3 20 2 5 1 218 1.04 14 8 3 1 29 4 6 1 1 19 0.98 36 61 14 7 34 4 8 1 2 1.05 133 18068 16 31 1 10 0 4 20 2.37 474 308 215 70 46 2 14 0 7 HER2 21 1.19 14 4 53 34 3 9 1 2 22 2.12 26 37 11 6 29 4 6 3 3 23 3.52 124 77 35 16 26 4 5 24 OZF, AKT2 24 1.59 732 396 282 65 29 3 7 1 5 25 1.26 26 31 10 1 40 3 111 6 1.34 129 138 57 19 46 3 13 0 7 AIB1, STK15 26 1.04 5 7 8 2 23 3 5 33 27 0.93 12 7 5 2 26 3 6 1 1 Loss and Deletion 28 −0.94 14 7 2 1 46 412 1 2 −0.73 151 67 64 18 49 3 14 1 1 29 −0.77 28 28 17 7 37 3 10 1 2−0.87 28 29 10 4 37 3 10 1 2 30 −1.69 5 1 0 0 49 4 13 1 5 31 −0.85 0 0 11 26 4 6 2 1 32 −1.03 0 12 1 0 23 2 6 1 1 33 −1 13 15 6 3 34 4 8 0 2 34−0.93 18 39 6 2 51 4 14 0 4 −0.91 24 40 14 0 51 4 14 1 4 35 −1.28 94 19440 8 54 6 13 0 2 36 −1 7 24 3 1 20 1 6 1 1 37 −1.11 133 181 56 21 51 513 0 6 38 −0.91 98 144 38 8 46 5 11 1 4 39 −0.72 22 62 8 2 46 5 11 1 340 −1.31 27 57 12 4 54 2 17 0 11 FEZ1 −0.95 60 131 28 16 46 1 15 0 11NRG1 41 −1.38 15 38 8 3 60 8 13 2 9 −2.63 129 216 55 15 57 7 13 1 11INK4A 42 −0.9 23 36 13 0 6 1 1 1 1 43 −0.73 123 129 68 18 29 2 8 1 1 44−0.73 116 70 70 19 34 3 9 1 1 −0.69 82 69 60 4 34 2 10 1 1 45 −0.8 57 9634 13 31 3 8 1 1 46 −1.19 63 111 27 9 37 4 9 2 4 DUSP6 47 −0.67 34 17 157 29 1 9 1 2 48 −0.99 18 34 14 0 57 5 15 1 5 TP53, MKK4 49 −1.19 21 9416 5 54 6 13 0 7 50 −1.53 98 287 23 8 60 7 14 0 7 51 −1.31 55 9 12 2 314 7 1 2 52 −1.24 2 8 3 0 60 5 16 1 2 53 −0.9 4 3 2 2 34 5 7 0 3 −0.87 81 6 1 40 5 9 0 3 54 −1.32 424 350 243 65 54 5 14 0 7

TABLE 2 List of cell lines and corresponding references used in thisstudy Name Source Reference PA-TU-8988T DSMZ Elsasser et al. VirchowsArch B Cell Pathol 61(5): 295-306. 1992 PA-TU-8988S DSMZ Elsasser et al.Virchows Arch B Cell Pathol 61(5): 295-306. 1992 PA-TU-8902 DSMZElsasser et al. Virchows Arch B Cell Pathol. 64: 201-207. 1993 DAN-GDSMZ Not Published HUP-T4 DSMZ Nishimura et al., Int. J. Pancreatol 13:31-41. 1993 HUP-T3 DSMZ Nishimura et al., Int. J. Pancreatol 13: 31-41.1993 Panc 10.05 ATCC Jaffee E M et al. Cancer J Sci Am 4: 194-203, 1998.PL45 ATCC Jaffee E M et al. Cancer J Sci Am 4: 194-203, 1998. Aspc-1ATCC Chen et al. In Vitro. 1982 January; 18(1): 24-34. Mpanc-96 ATCCPeiper M et al. Int. J. Cancer 71: 993-999, 1997. BxPC-3 ATCC Tan M H etal. Cancer Invest. 4: 15-23, 1986. Capan-1 ATCC Fogh et al. JNCI 58:209-214. 1977 Capan-2 ATCC Fogh et al. JNCI 58: 209-214. 1977 CFPAC-1ATCC Schoumacher et al. PNAS 87: 4012-4016. 1990 HPAF-II ATCC Kim Y W etal. Pancreas 4: 353-362. 1989. Hs766T ATCC Owens R. B. et al. JNCI 56:843-849, 1976. Panc-1 ATCC Lieber M et al. Int J. Cancer 15: 741-747.1975 SW1990 ATCC Kyriazis A P et al. Cancer Res. 43: 4393-4401. 1983 MIAPaCa-2 ATCC Yunis A A et al. Int. J. Cancer 19: 128-135. 1977 HPAC ATCCGower W R et al. In vitro Cell Dev Biol. 30A: 151-161. 1994 Panc 02.03ATCC Jaffee E M et al. Cancer J Sci Am 4: 194-203, 1998. Panc 02.13 ATCCJaffee E M et al. Cancer J Sci Am 4: 194-203, 1998. Panc 3.27 ATCCJaffee E M et al. Cancer J Sci Am 4: 194-203, 1998. Panc 08.13 ATCCJaffee E M et al. Cancer J Sci Am 4: 194-203, 1998.

TABLE 3 List of High and Moderal Confidence MCRs. Locus Locus PeakProfile Minimal Common Regions (MCRs) Cytogenetic Boundary Max/ Size Max# Trspt # Band (Mb) Pos (MB) Min Position (MB) (MB) Min K P 1 1p13.1-p12115.53-119.49 119.07-119.2  1.55 116.83-119.49 2.65 1.55 23 47 22p11.2-p11.1 85.23-91.48 85.78-85.78 1 85.75-85.85 0.1 1 10 5 33p11.1-q12.3  87.99-102.65  95.13-102.58 0.93 90.13-99.58 9.45 0.93 1837 101.35-101.83 0.48 0.93 5 7 4 5p15.33-p15.31 0.28-6.69 0.28-0.51 0.790.28-6.69 6.4 1.05 57 164 5 5q23.1-q31.1 115.81-132.47 131.44-132.271.08 115.81-132.47 16.66 1.08 110 182 6 5q31.1-q31.1 133.51-134.33133.56-133.95 0.97 133.53-133.56 0.04 0.97 0 0 7 5q31.3-q31.3139.21-140.22 139.48-140.22 0.65 139.21-140.04 0.83 0.65 39 16 98p22.1-p21.32 28.12-32.72 31.98-32.44 1.36 31.98-32.11 0.13 1.36 15 332.19-32.21 0.02 1.36 0 0 10 8p21.1-p21.1 42.91-43.19 42.98-43.15 0.8442.91-43.03 0.12 0.84 16 6 11 8q24.3-q25.1 145.88-151.63 150.09-151.191.45 150.09-151.12 1.03 1.45 16 32 12 7p22.3-p22.1 0.72-4.53 0.72-2.480.93 0.72-2.28 1.56 0.93 45 53 13 7p21.3-p21.2  7.39-14.44  7.97-14.440.58  7.39-14.44 7.04 0.8 20 58 14 7p15.1-p14.3 30.12-31.56  30.5-30.810.82 30.12-31.56 1.44 0.82 23 25 15 7p13-p11.2 44.93-54.88 43.64-47.711.23 44.93-54.88 9.95 1.23 41 143 16 7p11.21-q21.11 64.54-77.1864.95-64.95 1.06 64.95-65.85 0.9 1.06 12 22 17 7q21.11-q32.2 79.45-129.46 97.86-98.55 3.06  92.33-112.27 19.94 3.06 356 282 187q34-q38.1  142.7-150.22 143.26-143.38 0.94 143.38-147.82 4.44 0.94 6 3619 8p23.1-p22  8.75-12.74 11.05-11.57 2.05  8.76-12.74 3.98 2.05 43 7420 8p12-p11.21  37.7-41.76 37.72-38.02 1.71  37.7-38.45 0.75 1.71 31 1438.68-39.52 0.84 1.44 3 4 21 8q12.1-q12.3 59.09-63.66 59.23-62.26 0.5859.23-60.82 1.6 0.58 1 15 22 8q12.3-q13.1  63.7-67.06 65.44-66.81 0.87 63.7-67.06 3.37 0.87 20 43 23 8q21.3-q24.3  90.7-145.83 133.72-134.161.78 118.97-145.83 26.86 1.78 317 461 24 8p21.3-p13.2 23.68-37.8735.65-36.56 0.86  35.8-36.56 0.96 0.86 39 23 25 10q25.2-q25.3 112.4-113.62  112.4-112.77 0.85  112.4-115.62 3.22 0.85 20 61 2610q26.13-q28.2 124.92-129.83 126.14-126.14 0.65 124.92-126.41 1.49 0.659 30 27 11p12-q13.6 40.18-77.26    77-77.15 * 0.9 60.11-61.14 1.03 0.8132 19 28 11q14.1-q14.2 78.15-86.74 82.76-85.89 0.9 83.39-86.21 2.82 0.918 26 29 12p13.33-p13.2  0.18-11.45 0.18-2.79 1.77 0.18-0.74 0.56 1.7710 6 5.03-6.85 1.82 1.69 56 31 7.05-7.18 0.13 0.84 5 3 30 12p13.2-p13.111.95-13.06 12.18-12.77 0.6 11.95-13.06 1.11 0.96 26 23 3112p12.3-q13.13  16.6-53.06  20.7-22.54 2.52 21.82-22.39 0.57 2.52 9 524.67-31.14 6.27 1.49 71 91 32 12q14.3-q14.3 64.61-66.08 65.95-68.080.93 64.61-66.08 1.46 0.93 9 19 33 12q15-q15 68.27-68.87 68.44-68.771.38 68.27-68.85 0.59 1.38 15 13 34 12p24.11-q24.12 108.06-110.72108.55-110.68 0.98 108.06-110.72 2.66 0.98 59 37 35 12q24.31-q24.33120.76-133.4  123.83-125.12 1.5 122.12-131.01 8.89 1.5 123 132 3613q12.11-q14.15 18.68-43.95 19.21-24.22 0.89 18.68-19.21 0.53 0.89 6 620.65-24.85 4.19 0.89 43 74 37 13q14.2-q14.2 45.56-47.81 47.45-47.770.56 45.64-47.81 2.18 0.63 12 29 38 13q34-q34 112.84-113.06112.85-113.01 0.62 112.84-113.06 0.23 1.04 14 8 39 14q11.2-q24.318.82-74.47  73.56-74.47 * 1.08 28.12-33.17 5.05 0.98 36 61 48.08-51.173.09 0.74 61 40 52.41-63.53 11.12 1.05 133 180 40 14q24.3-q32.1175.23-88.86 75.25-86.96 0.93 75.23-87.87 12.63 0.93 65 138 4114p32.12-q32.33  91.16-104.37  91.39-104.37 0.63 103.33-103.61 0.28 0.6314 7 42 18p13.3-p13.3 0.03-4.95 3.37-3.49 1.23 1.81-1.95 0.15 0.85 7 53.27-3.34 0.26 1.23 13 13  3.9-4.52 0.62 0.94 11 18 43 16p13.3-p13.11 5.14-15.76  7.63-15.08 0.93 14.73-15.62 0.88 0.93 18 12 4415p12.2-p12.1 23.7-27.3  24.03-25.16 * 1.24 23.7-27.3 3.61 1.24 41 48 4517q11.2-2q11.2 29.14-30.78 30.22-30.66 0.74  30.5-30.78 0.28 0.74 7 3 4617q12-q23.2 37.48-56.39 43.53-43.53 2.37 38.95-55.26 16.31 2.37 474 30847 17q23.2-q25.3 59.83-79.64  82.33-62.47 * 3.61 74.02-74.22 0.2 1.19 144 48 18p11.21-q12.1 12.02-28.55 19.67-21.37 2.12 19.12-21.37 2.25 2.1226 37 49 19p13.11-q13.32 23.32-50    45.03-45.39 * 3.84 41.41-44.6  3.23.52 124 77 50 19q13.32-q13.43 50.06-63.76 50.85-50.65 1.59 50.06-62.8912.83 1.59 732 396 51 20p13-q13.33  0.33-63.41 58.54-63.41 1.3425.68-31.48 5.8 1.25 26 31 50.87-55.58 4.71 1.18 15 69 58.24-63.41 5.171.34 129 138 52 22q11.1-q12.1 14.65-26.58 22.7-22.7 1.04 22.64-22.83 0.21.04 5 7 53 22q12.2-q12.3 28.75-29.85 28.96-29.83 0.93 29.35-29.85 0.510.93 12 7 54 1p38.21-p36.11 15.02-26.95 21.46-21.46 −0.94 21.08-21.560.48 −0.94 14 7 22.82-26.95 4.13 −0.73 151 67 55 1p35.3-p34.328.37-39.18  32.79-33.04 * −0.87 28.37-31.18 2.81 −0.77 28 2832.67-34.68 2.01 −0.87 28 29 56 1p34.2-p34.2 40.39-42.59 42.09-42.11−0.75 40.39-42.59 2.21 −0.75 32 28 57 1p32.2-p31.3 56.74-60.9556.77-59.75 −0.59 56.74-60.95 4.21 −0.67 17 46 58 1p21.2-p21.1100.63-103.3  103.25-103.25 1.69 103.25-103.3 0.05 −1.69 5 1 592p25.3-p24.3  0.21-16.09  10.92-11.3 * −0.85 0.21-7.04 6.83 −0.73 36 146 11.3-11.32 0.02 −0.85 0 0 60 2p12-p11.2 75.23-89    79.31-85.51 * −1.03 77.7-79.27 1.57 −1.03 0 12 86.97-89   2.03 −0.78 15 37 61 3p26.3-p24.31.33-18.8 13.65-14.52 −1 11.29-12.18 0.89 −0.78 8 6 13.57-14.52 0.85 −113 15 62 3p21.32-p14.1 44.51-66.3  53.69-57.41 −0.87 53.08-61.58 8.51−0.87 68 122 63 4p16.3-p16.2 4.27-4.86 4.41-4.43 −0.82 4.27-4.86 0.59−0.82 3 11 64 4p15.32-p15.2 17.28-26.18 24.69-26.12 −0.94 17.28-26.188.91 −0.94 49 107 65 4q13.2-q13.3 69.37-72.84 69.37-71.59 −0.6469.37-72.84 3.47 −0.65 47 56 66 4q22.1-q22.1 88.79-89.29 89.13-89.13−0.75 88.79-89.29 0.5 −0.75 5 8 67 4q24-q24 104.07-103.95 104.07-104.07−0.62 104.07-103.95 −0.13 −0.62 0 0 68 4q27-q28.1 123.22-129.09123.77-124.24 −0.75 123.22-129.09 5.87 −0.75 27 49 69 4q31.22-q32.1148.03-158.75  149.46-153.1 * −0.93 151.71-154.37 2.65 −0.93 18 39155.09-156.56 3.47 −0.91 24 40 70 4q34.1-q35.2 174.84-188.09175.03-187.79 −1.28 174.84-188.09 13.26 −1.28 94 194 71 5q23.2-q23.3127.55-130.53 127.65-128.48 −0.62 127.55-130.53 2.98 −1 7 24 726p25.3-p12.2  0.4-52.14  0.4-2.83 −1.17  0.4-2.83 2.42 −1.17 14 50 26.1-27.86 1.76 −0.86 75 55 73 6p12.1-p12.1  56.4-57.04 56.4-56.4 −0.64 56.4-57.04 0.64 −0.72 10 7 74 6q21-q22.31 106.63-119.43 107.02-116.57−1.11 106.63-119.43 12.8 −1.11 133 181 75 6q23.3-q24.3 135.17-146.81137.46-138.13 −0.91 135.17-146.81 11.64 −0.91 98 144 76 6q26.3-q27180.39-165.66 160.51-162.94 −0.73 160.39-165.66 5.27 −0.73 37 110 776q27-q27 168.07-170.54 168.63-170.54 −0.72 168.07-170.54 2.47 −0.72 2282 78 7p21.2-p21.1 14.44-18.87 17.08-18.86 −0.9 14.44-18.87 4.43 −0.9 1743 79 7q34-q36.1 142.47-147.82 143.2-143.38 −0.8  142.7-147.82 5.12 −0.817 83 80 8p23.3-p12 2.06-37.7  2.06-2.1 * −1.85 18.07-21.75 3.68 −1.3127 87 28.45-37.7  9.25 −0.95 60 131 81 8p12-p11.21  37.7-42.4538.45-42.33 −1.19  37.7-39.05 1.35 −1.19 41 21 82 9p24.3-p21.2 0.47-27.18 20.77-21.31 −2.53 0.47-3.39 2.92 −1.38 15 38  5.3-23.6818.39 −2.53 129 215 83 9q13-q21.11 65.19-66.51 65.26-65.51 −1.2765.19-66.51 1.32 −1.27 11 21 84 9q22.33-q31.1  97.63-101.94 97.64-101.81 −0.81  97.63-101.94 4.31 −0.81 31 44 85 9q31.2-q31.3105.59-109.38 106.59-107.89 −1 108.59-108.63 2.04 −1 22 19 869q34.11-q34.11 127.73-128.53 127.75-127.81 −0.91 127.73-128.53 0.8 −0.9117 9 87 10p15.3-p15.3 0.29-1.17 0.29-1.08 −0.81 0.29-1.17 0.88 −0.78 1221 88 10p12.33-p12.1 17.91-26.66 18.09-18.32 −3.3 17.91-26.66 8.75 −3.348 110 89 10p12.1-p11.22 27.19-33.36 27.63-32.71 −0.62 27.19-33.36 6.17−0.78 63 94 90 10q22.1-q22.1 73.46-73.72 73.46-73.47 −3.23 73.46-73.720.26 −3.23 2 4 91 10q22.3-q26.3  81.01-135.27 126.41-135.27 −0.93126.14-135.27 9.13 −0.93 92 204 92 11p15.4-p15.3 10.84-13   12.02-12.02−3.72 10.84-13   2.16 −3.72 24 35 93 11p15.2-p15.1 14.58-18.4717.38-18.43 −0.92 14.58-18.47 3.89 −0.92 38 49 94 11p15.1-p1320.11-32.17 20.42-31.85 −1.2 20.11-32.17 12.05 −1.2 80 100 9511q14.1-q14.2 78.15-85.59 82.76-83.39 −0.67 78.15-85.59 7.44 −0.67 25 5596 11q14.2-q14.3 85.89-89.24 86.21-89.24 −0.9 85.89-59.24 3.35 −0.9 2336 97 11q21-q23.2  95.74-114.13  95.74-114.13 −0.75  95.74-114.13 18.39−0.75 188 207 98 11q23.5-q25 116.73-134.28  117.1-134.27 −0.7120.33-121.54 1.21 −0.7 10 19 99 12q12-q13.12 40.06-49.24 41.04-49.21−0.71 40.06-49.24 9.18 −0.73 123 129 100 12q13.12-q13.3 49.65-55.8250.01-53.06 −0.7 49.65-53.06 3.41 −0.73 118 70 53.17-55.82 2.65 −0.69 8269 101 12q14.1-q15 57.92-68.77 62.76-68.27 −0.8 62.76-68.77 6.01 −0.8 5796 102 12q21.2-q24.33 77.19-133.4 81.03-85.63 −1.19 77.19-91.44 14.25−1.19 63 111 103 14q24.2-q24.3 71.68-72.03 71.72-71.73 −1.1 71.68-72.030.35 −1.1 5 6 104 16p13.3-p12.2  0.03-23.97 0.76-23.7 −0.67 2.24-2.820.58 −0.57 34 17 105 16q12.1-q12.2 48.37-55.26 50.08-53.88 −1.148.37-53.88 5.52 −1.1 35 83 106 17p13.3-q11.1  0.02-25.81  8.16-14.05−0.99 10.36-12.8  2.44 −0.99 18 34 107 17q11.2-q11.2 26.68-30.2226.68-28.65 −0.83  27.8-28.97 1.17 −0.83 17 12 108 17q24.2-q25.366.73-75.58 66.98-71.86 −0.92 71.63-71.86 0.23 −0.92 10 6 11018q11.2-q21.1 18.51-48.28 34.95-40.58 −1.19 34.16-43.14 8.99 −1.19 21 94111 18q22.1-q23  60.4-77.63 74.45-76.84 −1.53  60.4-77.63 17.23 −1.53 96257 112 19p13.3-p13.2 4.62-9.11 6.63-6.68 −0.86 6.61-6.85 0.24 −0.88 114 113 19p13.2-p13.2 12.83-13.16 12.84-13.11 −0.88  12.9-13.07 0.17 −0.8810 3 114 19q13.2-q13.43 44.57-63.76 59.99-60.04 −1.31 50.65-51.57 0.92−0.68 40 29 59.85-60.18 0.33 −1.31 55 9 115 20p13-q11.21  0.33-30.89 8.81-25.63 −1.39  5.91-25.68 19.77 −1.39 139 321 116 21p11.2-q11.2 9.96-13.43  9.96-10.08 −1.24  9.96-13.43 3.47 −1.24 2 8 11721q22.11-q22.11 31.95-32.5  32.16-32.5  −1.15 31.95-32.5  0.55 −1.10 3 8118 21q22.11-q22.12 34.19-36.62 35.08-35.42 −1.09 34.01-35.42 0.61 −1.095 14 119 21q22.13-q22.13 37.01-37.52 37.01-37.02 −1.58 37.01-37.52 0.5−1.58 9 10 120 21q22.2-q22.3 39.76-46.94  41.7-41.75 * −4.09 45.08-45.170.09 −0.9 4 3 46.77-46.94 0.17 −0.87 8 1 121 22q11.1-q13.2 14.49-39.4622.7-22.7 −1.32 20.64-39.46 18.83 −1.32 424 350 Minimal Common Regions(MCRs) MCR Recurrence Affu # on U133A Gain/Loss Amp/Del genes Affu #Total Sig. % T C T C signif genes ID CNA Bands 1 15 2 23 2 6 2 2 2 15 291 1p13.1-1p12 2 7 2 14 0 5 0 2 2 7 2 2 2p11.2-2p11.1 3 5 0 29 1 9 0 2 05 31 3 3p11.1-3q12.3 5 5 17 0 6 0 2 5 5 32 3 3p11.1-3q12.3 4 19 8 43 114 0 5 8 19 5 4 5p15.33-5p15. 5 44 6 26 1 8 0 1 6 44 77 5 5q23.1-5q31.16 1 0 14 1 4 1 1 0 1 78 6 5q31.1-6q31.1 7 10 0 31 2 9 0 2 0 10 113 75q31.3-5q31.3 9 14 1 29 1 9 0 4 1 14 34 8 8p22.1-6p21.3 2 0 29 1 9 0 3 02 35 9 8p22.1-6p21.3 10 7 2 23 2 6 1 1 2 7 125 10 6p21.1-8p21.1 11 4 117 0 6 0 2 1 4 8 11 8q24.3-8q25.1 12 10 6 51 4 14 0 7 6 10 36 127p22.3-7p22.1 13 13 3 40 2 12 1 2 3 13 155 13 7p21.3-7p21.2 14 13 1 37 112 1 2 1 13 126 14 7p13.1-7p14.3 15 27 13 34 0 12 0 2 13 27 62 157p13-7p11.2 16 7 5 34 1 11 1 1 5 7 9 16 7q11.21-7q21. 17 166 42 46 4 121 5 42 166 11 17 7q21.11-7q32. 18 4 0 17 0 6 0 2 0 4 38 18 7q34-7q36.119 14 3 11 0 4 0 1 3 14 63 19 8p23.1-8p22 20 14 8 37 1 12 0 2 6 14 64 208p12-8p11.21 5 0 31 1 10 0 2 0 5 65 20 8p12-8p11.21 21 4 0 49 4 13 1 1 04 96 21 8q12.1-8q12.3 22 6 0 43 3 12 0 1 0 6 127 22 8q12.3-8q13.1 23 11541 56 6 17 0 7 41 115 13 23 8q21.3-8q24.3 24 18 9 20 1 6 0 3 9 18 14 249p21.3-9p13.2 25 15 3 26 2 7 0 1 3 15 15 25 10q25.2-10q2 26 7 0 23 1 7 02 0 7 74 26 10q26.13-10q2 27 13 0 40 1 13 0 3 0 13 16 27 11p12-11q13.528 9 0 46 0 16 0 2 0 9 87 28 11q14.1-11q14 29 4 1 23 2 6 0 2 1 4 99 2912p13.33-12p3 33 14 20 1 6 0 2 14 33 100 29 12p13.33-12p3 2 1 20 1 6 0 21 2 101 29 12p13.33-12p3 30 14 8 26 3 6 0 2 8 14 68 30 12p13.2-12p13 313 1 26 2 7 2 2 1 3 40 31 12p12.3-12q13 26 13 34 1 11 0 3 13 26 41 3112p12.3-12q13 32 7 0 17 1 5 0 2 0 7 42 32 12q14.3-12q14 33 1 0 23 2 6 12 0 1 114 33 12q15-12q15 34 28 0 20 1 6 0 3 9 28 88 34 12q24.11-12q2 3529 0 20 1 6 0 3 9 29 89 35 12q24.31-12q2 36 3 0 20 2 5 0 2 0 3 81 3613q12.11-13q2 19 3 20 2 5 1 2 3 19 82 36 13q12.11-13q2 37 12 4 26 4 5 12 4 12 90 37 13q14.2-13q14 38 3 1 29 4 6 1 1 1 3 67 38 13q34-13q34 39 147 34 4 8 1 2 7 14 18 39 14q11.2-14q24 26 17 29 2 8 0 3 17 26 19 3914q11.2-14q24 68 16 31 1 10 0 4 16 68 20 39 14q11.2-14q24 40 29 9 17 0 60 2 9 29 75 40 14q24.3-14q32 41 7 4 23 3 5 0 3 4 7 43 41 14q32.12-14q342 4 2 11 1 3 0 2 2 4 21 42 16p13.3-16p13 9 3 14 0 5 0 2 3 9 22 4216p13.3-16p13 6 2 14 0 5 0 2 2 6 23 42 16p13.3-16p13 43 5 2 20 1 6 0 2 25 24 43 16p13.3-16p13 44 0 4 26 2 7 1 0 4 9 148 44 16p12.2-16p12 45 1 026 3 6 1 1 0 1 142 45 17q11.2-17q12 46 215 70 46 2 14 0 7 70 215 27 4617q12-17q23.2 47 5 3 34 3 9 1 2 3 5 92 47 17q23.2-17q25 48 11 6 29 4 6 33 6 11 140 48 18p11.21-18q 49 35 16 26 4 5 2 4 18 35 45 49 19p13.11-19q50 282 65 29 3 7 1 5 65 282 46 50 19q13.32-19q 51 10 1 40 3 11 1 5 1 1047 51 20p13-20q13.3 12 4 46 3 13 0 7 4 12 48 51 20p13-20q13.3 57 19 46 313 0 7 19 57 49 51 20p13-20q13.3 52 6 2 23 3 5 3 3 2 6 83 5222q11.1-22q12 53 5 2 26 3 6 1 1 2 5 115 53 22q12.2-22q12 54 2 1 46 4 122 2 1 2 236 54 1p36.21-1p36. 64 18 49 3 14 1 1 18 84 237 541p36.21-1p36. 55 17 7 37 3 10 1 2 7 17 182 55 1p35.3-1p34.3 10 4 37 3 101 2 4 10 183 55 1p35.3-1p34.3 56 14 4 29 3 7 0 0 4 14 283 561p34.2-1p34.2 57 7 2 37 3 10 1 2 2 7 252 57 1p32.2-1p31.3 58 0 0 49 4 131 5 0 0 238 58 1p21.2-1p21.1 59 14 6 34 3 9 1 3 6 14 161 592p25.3-2p24.3 1 1 26 4 5 2 1 1 1 162 59 2p25.3-2p24.3 60 1 0 23 2 6 1 10 1 230 60 2p12-2p11.2 10 0 11 1 3 1 1 0 10 231 60 2p12-2p11.2 61 5 2 345 7 1 1 2 5 163 61 3p26.3-3p24.3 5 3 34 4 8 0 2 3 8 164 61 3p26.3-3p24.362 25 12 43 3 12 0 4 12 28 233 62 3p21.32-3p14 63 2 1 37 4 0 0 2 1 2 18563 4p16.3-4p16.2 64 15 5 40 4 10 0 4 5 15 239 64 4p15.32-4p15 65 28 4 514 14 0 2 4 28 265 65 4q13.2-4q13.3 66 6 0 48 3 13 0 2 0 8 218 664q22.1-4q22.1 67 0 0 43 2 13 0 2 0 0 219 67 4q24-4q24 68 9 2 48 3 13 0 22 9 165 68 4q27-4q28.1 69 8 2 51 4 14 0 4 2 6 167 69 4q31.22-4q32. 14 051 4 14 1 4 0 14 168 69 4q31.22-4q32. 70 40 8 54 5 13 0 2 8 40 210 704q34.1-4q35.2 71 3 1 20 1 8 1 1 1 3 281 71 5q23.2-5q23.3 72 7 1 40 6 5 04 1 7 184 72 6p25.3-5p12.2 56 7 29 4 5 0 3 7 56 185 72 6p25.3-6p12.2 736 1 34 5 7 1 3 1 5 279 73 6p12.1-6p12.1 74 66 21 51 6 13 0 6 21 56 18774 6q21-6q22.31 75 38 8 48 5 11 1 4 5 38 241 75 6q23.3-6q24.3 76 18 6 485 11 0 5 8 18 188 76 6q25.3-6q27 77 4 2 48 5 11 1 3 2 8 282 77 6q27-6q2778 13 1 9 1 2 1 0 1 13 295 78 7p21.2-7p21.1 79 12 0 31 3 8 2 2 0 12 26579 7q34-7q36.1 80 12 4 54 2 17 0 11 4 12 190 80 8p23.3-8p12 28 16 48 115 0 11 18 28 191 80 8p23.3-8p12 81 17 3 37 1 12 0 5 3 17 212 818p12-8p11.21 82 8 3 60 6 13 2 9 3 8 170 82 9p24.3-9p21.2 55 15 57 7 13 111 18 65 171 82 9p24.3-9q21.2 83 0 0 40 5 9 0 2 0 0 272 83 9q13-9q21.1184 13 2 14 2 3 1 0 2 13 297 84 9q22.33-9q31. 85 13 8 14 1 4 0 2 8 13 25085 9q31.2-9q31.3 86 10 5 14 2 3 1 0 5 10 285 86 9q34.11-9q34 87 7 3 40 311 1 4 3 7 258 87 10p15.3-10p15 88 24 4 57 1 12 0 5 4 24 173 8810p12.33-10p 89 20 9 34 2 10 1 4 9 20 298 89 10p12.1-10p 90 1 0 17 1 5 02 0 1 174 90 10q22.1-10q22 91 34 16 26 2 7 0 2 15 34 221 9110q22.3-10q26 92 10 4 25 1 5 0 2 4 10 175 92 11p15.4-11p15 93 27 4 29 19 0 3 4 27 195 93 11p15.2-11p15 94 23 7 28 1 8 0 2 7 23 198 9411p15.1-11p13 95 11 0 8 1 1 1 1 0 11 204 95 11q14.1-11q14 96 13 0 8 1 11 1 0 13 222 96 11q14.2-11q14 97 75 8 17 2 4 1 1 8 75 223 9711q21-11q23.2 98 4 1 11 1 3 1 2 1 4 178 98 11q23.3-11q25 99 58 18 29 2 81 1 18 55 290 99 12q12-12q13. 100 70 19 34 3 9 1 1 19 70 291 10012q13.12-12q 60 4 34 2 10 1 1 4 80 292 100 12q13.12-12q 101 34 13 31 3 81 1 13 34 293 101 12q14.1-12q15 102 27 9 37 4 9 2 4 9 27 197 10212q21.2-12q2 103 2 1 17 2 4 1 0 1 2 294 103 14q24.2-14q2 104 15 7 29 1 91 2 7 15 242 104 16p13.3-16p12 105 24 6 11 1 3 0 2 8 24 192 10516q12.1-16q12 106 14 0 57 5 15 1 5 0 14 178 106 17p13.3-17q1 107 5 1 263 6 0 2 1 5 225 107 17q11.2-17q1 108 2 0 23 2 6 0 3 0 2 228 10817p24.2-17q25 110 16 5 54 6 13 0 7 5 16 208 110 18q11.2-18q2 111 23 8 507 14 0 7 8 23 213 111 18q22.1-18q23 112 5 2 31 2 9 0 2 2 6 196 11219p13.3-19p13 113 4 3 29 2 8 0 2 3 4 158 113 19p13.2-19p13 114 21 12 344 8 1 2 12 21 159 114 19q13.2-19q13 12 2 31 4 7 1 2 2 12 150 11419q13.2-19q13 115 76 27 34 2 10 0 6 27 75 194 115 20p13-20q11.3 116 3 060 5 18 1 2 0 3 200 116 21p11.2-21q1 117 5 0 34 3 9 0 2 0 3 208 11721q22.11-21q3 118 4 0 40 4 10 0 3 0 4 214 118 21q22.11-21q3 119 5 2 37 49 0 1 2 5 215 119 21q22.15-21q3 120 2 2 34 5 7 0 3 2 2 243 12021q22.2-21q23 6 1 40 5 9 0 3 1 6 244 120 21q22.2-21q23 121 243 85 54 514 0 7 65 243 179 121 22q11.1-22q13 # Locus Max.Min Max:Min.Val MCR.LocMCR.Size 1 TRUE 116.83-119.49 119.07-119.2 1.55 118.83-119.49 2.65 2TRUE 85.23-91.48 95.78-85.78 1 85.75-85.85 0.1 3 TRUE 87.99-102.6595.13-102.58 0.93 90.13-99.58 9.45 FALSE 87.99-102.65 95.13-102.58 0.93101.35-101.83 0.48 4 TRUE 0.28-6.69 0.28-0.51 0.79 0.28-6.69 6.4 5 TRUE115.81-132.47 131.44-132.27 1.08 115.81-132.47 16.66 6 TRUE133.51-134.33 133.56-133.95 0.97 133.53-133.56 0.04 7 TRUE 139.21-140.22139.48-140.22 0.65 139.21-140.04 0.83 9 TRUE 28.12-32.72 31.98-32.441.36 31.98-32.11 0.13 FALSE 28.12-32.72 31.98-32.44 1.36 32.19-32.210.02 10 TRUE 42.91-43.19 42.98-43.15 0.84 42.91-43.03 0.12 11 TRUE145.88-131.63 150.09-151.19 1.45 150.09-151.12 1.03 12 TRUE 0.72-4.530.72-2.48 0.93 0.72-2.28 1.56 13 TRUE  7.39-14.44  7.97-14.44 0.58 7.39-14.44 7.04 14 TRUE 30.12-31.56  30.5-30.81 0.82 30.12-31.56 1.4415 TRUE 44.93-54.88 45.64-47.71 1.23 44.93-54.88 9.95 16 TRUE64.84-77.16 64.95-84.95 1.06 64.95-65.85 0.9 17 TRUE  79.45-129.4697.86-98.55 3.06  92.33-112.27 19.94 18 TRUE  142.7-150.22 143.26-143.380.94 143.38-147.82 4.44 19 TRUE  8.76-12.74 11.05-11.57 2.05  8.76-12.743.98 20 TRUE  37.7-41.76 37.72-38.02 1.71  37.7-38.45 0.75 FALSE 37.7-41.76 37.72-38.02 1.71 38.68-39.52 0.84 21 TRUE 59.09-63.6650.23-62.26 0.58 59.23-60.82 1.6 22 TRUE  63.7-67.06 65.44-66.81 0.87 63.7-67.06 3.37 23 TRUE  90.7-145.83 133.72-134.16 1.78 118.97-145.8326.86 24 TRUE 23.68-37.87 35.65-36.56 0.86  35.6-36.56 0.96 25 TRUE 112.4-115.62  112.4-112.77 0.85  112.4-115.62 3.22 26 TRUE124.92-129.83 126.14-126.14 0.65 124.92-126.41 1.49 27 TRUE 40.18-77.26  77-77.15 0.9 60.11-31.14 1.03 28 TRUE 78.15-86.74 82.76-85.89 0.983.39-86.21 2.82 29 TRUE  0.18-11.45 0.18-2.79 1.77 0.18-0.74 0.56 FALSE 0.18-11.45 0.18-2.79 1.77 5.03-6.85 1.82 FALSE  0.18-11.45 0.18-2.791.77 7.05-7.18 0.13 30 TRUE 11.95-13.06 12.18-12.77 0.6 11.95-13.06 1.1131 TRUE  16.6-53.06  20.7-22.54 2.52 21.82-22.39 0.57 FALSE  16.6-53.06 20.7-22.54 2.52 24.87-31.14 6.27 32 TRUE 64.61-66.08 65.95-66.06 0.9364.61-66.08 1.46 33 TRUE 68.27-68.87 68.44-68.77 1.38 68.27-68.85 0.5934 TRUE 108.06-110.72 108.55-110.68 0.98 108.06-110.72 2.66 35 TRUE120.76-133.4  123.83-125.12 1.5 122.12-131.01 8.89 36 TRUE 18.68-43.9519.21-24.22 0.89 18.68-19.21 0.53 FALSE 18.68-43.95 19.21-24.22 0.8920.65-24.85 4.19 37 TRUE 47.56-47.81 47.45-47.77 0.58 45.64-47.81 2.1838 TRUE 112.84-113.06 112.85-113.01 0.62 112.84-113.06 0.23 39 TRUE18.82-74.47 73.56-74.47 1.08 28.12-33.17 5.05 FALSE 18.82-74.4773.56-74.47 1.08 48.08-51.17 3.09 FALSE 18.82-74.47 73.56-74.47 1.0852.41-63.53 11.12 40 TRUE 75.23-88.86 75.25-86.96 0.93 75.23-87.87 12.6341 TRUE  91.16-104.37  91.39-104.37 0.63 103.33-103.61 0.28 42 TRUE0.03-4.95 3.37-3.49 1.23 1.81-1.95 0.15 FALSE 0.03-4.95 3.37-3.49 1.233.27-3.84 0.26 FALSE 0.03-4.95 3.37-3.49 1.23  3.9-4.52 0.62 43 TRUE 5.14-15.76  7.63-15.08 0.93 14.73-15.62 0.88 44 TRUE 23.7-27.324.03-25.16 1.24 23.7-27.3 3.61 45 TRUE 29.14-30.78 30.32-30.66 0.74 30.5-30.78 0.28 46 TRUE 37.48-56.39 43.53-43.53 2.87 38.95-55.26 16.3147 TRUE 59.83-79.64 62.33-62.47 3.61 74.02-74.22 0.2 48 TRUE 12.02-28.5519.67-21.37 2.12 19.12-21.37 2.25 49 TRUE 23.32-50   45.03-45.39 3.8441.41-44.6  3.2 50 TRUE 50.06-63.76 50.65-50.65 1.59 50.06-62.89 12.8351 TRUE  0.33-63.41 58.54-63.41 1.34 25.68-31.48 5.8 FALSE  0.33-63.4158.54-63.41 1.34 50.87-55.58 4.71 FALSE  0.33-63.41 58.54-63.41 1.3458.24-63.41 5.17 52 TRUE 14.65-28.58 22.7-22.7 1.04 22.64-22.83 0.2 53TRUE 28.75-29.85 28.96-29.83 0.93 29.35-29.85 0.51 54 TRUE 15.02-26.9621.46-21.46 −0.94 21.08-21.56 0.48 FALSE 15.02-26.95 21.46-21.46 −0.9422.82-26.95 4.13 55 TRUE 28.37-39.18 32.79-33.04 −0.87 28.37-31.18 2.81FALSE 28.37-39.18 32.79-33.04 −0.87 32.67-34.68 2.01 56 TRUE 40.39-42.5942.09-42.11 −0.75 40.39-42.59 2.21 57 TRUE 56.74-60.95 56.77-59.75 −0.5956.74-60.95 4.21 58 TRUE 100.63-103.3  103.25-103.25 −1.69 103.25-103.30.05 59 TRUE  0.21-16.09 10.92-11.3  −0.65 0.21-7.04 6.83 FALSE 0.21-16.09 10.92-11.3  −0.85 11.3-11.32 0.02 60 TRUE 75.23-89  79.31-85.51 −1.03  77.7-79.27 1.57 FALSE 75.23-89   79.31-85.51 −1.0386.97-89   2.03 61 TRUE 1.33-18.8 13.85-14.52 −1 11.29-12.18 0.89 FALSE1.33-18.8 13.65-14.52 −1 13.67-14.52 0.85 62 TRUE 44.51-66.3 53.69-57.41 −0.87 53.08-61.58 8.51 63 TRUE 4.27-4.86 4.41-4.43 −0.824.27-4.86 0.59 64 TRUE 17.28-25.18 24.69-26.12 −0.94 17.28-26.18 8.91 65TRUE 69.37-72.84 69.37-71.59 −0.64 69.37-72.84 3.47 66 TRUE 88.79-89.2989.13-89.13 −0.75 88.79-89.29 0.5 67 TRUE 104.07-103.95 104.07-104.07−0.62 104.07-103.95 −0.13 68 TRUE 123.22-129.09 123.77-124.24 −0.75123.22-129.09 5.87 69 TRUE 148.03-158.75 149.46-153.1 −0.93151.71-154.37 2.65 FALSE 148.03-158.75 149.46-153.1 −0.93 155.09-158.563.47 70 TRUE 174.84-188.09 175.03-187.79 −1.28 174.84-188.09 13.26 71TRUE 127.55-130.53 127.65-128.48 −0.62 127.55-130.53 2.98 72 TRUE 0.4-52.14  0.4-2.83 −1.17  0.4-2.83 2.42 FALSE  0.4-52.14  0.4-2.83−1.17  25.1-27.86 1.76 73 TRUE  56.4-57.04 56.4-56.4 −0.64  58.4-57.040.64 74 TRUE 106.63-119.43 107.02-116.57 −1.11 106.83-119.43 12.8 75TRUE 135.17-146.81 137.46-138.13 −0.91 135.17-146.81 11.64 76 TRUE160.39-165.66 160.51-162.94 −0.73 160.39-165.66 5.27 77 TRUE158.07-170.54 168.63-170.54 −0.72 168.07-170.54 2.47 78 TRUE 14.44-18.8717.08-18.86 −0.9 14.44-18.87 4.43 79 TRUE 142.47-147.82  143.2-143.36−0.8  142.7-147.82 5.12 80 TRUE 2.06-37.7 2.06-2.1  −1.86 18.07-21.753.68 FALSE 2.06-17.7 2.06-2.1  −1.86 28.45-37.7  9.25 81 TRUE 37.7-42.45 38.45-42.33 −1.19  37.7-39.05 1.35 82 TRUE  0.47-27.1820.77-21.31 −2.53 0.47-3.39 2.92 FALSE  0.47-27.18 20.77-21.31 −2.53 5.3-23.68 18.39 83 TRUE 65.19-66.51 65.26-65.51 −1.27 65.19-66.51 1.3284 TRUE  97.63-101.94  97.64-101.81 −0.81  97.63-101.94 4.31 85 TRUE105.59-109.38 106.59-107.89 −1 106.59-106.63 2.04 86 TRUE 127.73-128.53127.75-127.81 −0.91 127.73-128.53 0.8 87 TRUE 0.29-1.17 0.29-1.08 −0.610.29-1.17 0.88 88 TRUE 17.91-26.66 18.09-18.32 −3.3 17.91-26.66 8.75 89TRUE 27.19-33.36 27.63-32.71 −0.62 27.19-33.36 6.17 90 TRUE 73.46-73.7273.46-73.47 −3.23 73.46-73.72 0.26 91 TRUE  81.01-135.27 126.41-135.27−0.93 126.14-135.27 9.13 92 TRUE 10.84-13   12.02-12.02 −3.72 10.84-13  2.16 93 TRUE 14.58-18.47 17.38-18.43 −0.92 14.58-18.47 3.89 94 TRUE20.11-32.17 20.42-31.85 −1.2 20.11-32.17 12.05 95 TRUE 78.15-85.5982.76-83.39 −0.67 78.15-85.59 7.44 96 TRUE 85.89-89.24 86.21-89.24 −0.985.89-89.24 3.35 97 TRUE  95.74-114.13  95.74-114.13 −0.75  95.74-114.1318.39 98 TRUE 116.73-134.28  117.1-134.27 −0.7 120.33-121.54 1.21 99TRUE 40.06-49.24 41.04-49.21 −0.71 40.06-49.24 9.18 100 TRUE 49.55-55.8250.01-53.06 −0.7 49.65-53.06 3.41 FALSE 49.55-55.82 50.01-53.06 −0.753.17-55.82 2.65 101 TRUE 57.92-68.77 62.76-68.27 −0.8 62.76-68.77 6.01102 TRUE 77.19-133.4 81.03-85.63 −1.19 77.19-91.44 14.25 103 TRUE71.68-72.03 71.72-71.73 −1.1 71.68-72.03 0.35 104 TRUE  0.03-23.970.76-23.7 −0.67 2.24-2.82 0.58 105 TRUE 48.37-55.26 50.08-53.88 −1.148.37-53.88 5.52 106 TRUE  0.02-25.81  8.16-14.05 −0.99 10.36-12.8  2.44107 TRUE 25.68-30.32 26.68-28.65 −0.83  27.8-28.97 1.17 108 TRUE66.73-76.58 66.96-71.86 −0.92 71.63-71.86 0.23 110 TRUE 18.51-46.2834.95-40.58 −1.19 34.18-43.14 8.99 111 TRUE  60.4-77.63 74.45-76.84−1.53  60.4-77.63 17.23 112 TRUE 4.62-9.11 6.63-6.68 −0.86 6.61-6.850.24 113 TRUE 12.83-13.18 12.84-13.11 −0.88  12.9-13.07 0.17 114 TRUE44.57-63.76 59.99-60.04 −1.31 50.65-51.57 0.92 FALSE 44.57-63.7659.99-60.04 −1.31 59.85-60.18 0.33 115 TRUE  0.33-30.89  8.81-25.63−1.39  5.91-25.68 19.77 116 TRUE  9.96-13.43  9.96-10.08 −1.24 9.96-13.43 3.47 117 TRUE 31.95-32.5  32.16-32.5  −1.16 31.95-32.5  0.55118 TRUE 34.19-36.62 35.08-35.42 −1.09 34.81-35.42 0.61 119 TRUE37.01-37.52 37.01-37.02 −1.58 37.01-37.52 0.5 120 TRUE 39.76-46.94 41.7-41.75 −4.09 45.08-45.17 0.09 FALSE 39.76-46.94  41.7-41.75 −4.0946.77-46.94 0.17 121 TRUE 14.49-39.46 22.7-22.7 −1.32 20.64-39.46 18.83

TABLE 4 Markers of the invention which reside in MCRs of deletion anddisplay decreased expression. Pos Gene Minimum Chromosome (Mb) Weight pvalue Gene Description Gene Symbol GI UGID# 1 21.3 −0.84 0.005 alkalinephosphatase, liver/bone/kidney ALPL gi: 28737 Hs.250769 1 22.8 −1.12<.005 KIAA0601 protein KIAA0601 gi: 3043725 Hs.348515 1 23.3 −0.38 0.037E2F transcription factor 2 E2F2 gi: 4758225 Hs.231444 1 23.5 −0.73 <.005lysophospholipase II LYPLA2 gi: 9966763 Hs.413781 1 23.5 −0.39 0.049galactose-4-epimerase, UDP- GALE gi: 9945333 Hs.76057 1 23.6 −0.56 0.0123-hydroxymethyl-3-methylglutaryl-Coenzyme HMGCL gi: 4504426 Hs.444925 Alyase (hydroxymethylglutaricaciduria) 1 23.6 −0.80 <.005lysophospholipase II LYPLA2 gi: 4376011 Hs.413781 1 23.6 −0.42 0.03fucosidase, alpha-L-1, tissue FUCA1 gi: 4503802 Hs.576 1 24.3 −0.48<.005 serine/arginine repetitive matrix 1 SRRM1 gi: 5032118 Hs.18192 124.5 −0.64 0.021 chloride intracellular channel 4 CLIC4 gi: 4588523Hs.25035 1 24.9 −0.61 0.005 hypothetical protein dJ465N24.2.1DJ465N24.2.1 gi: 12005626 Hs.259412 1 25.8 −0.87 <.005 stathminl/oncoprotein 18 STMN1 gi: 13518023 Hs.209983 1 26.1 −0.45 0.037 zincfinger protein ZT86 gi: 7705661 Hs.102419 1 26.4 −0.44 0.021high-mobility group nucleosomal binding HMGN2 gi: 13277559 Hs.181163domain 2 1 26.4 −0.86 <.005 ribosomal protein S6 kinase, 90 kDa, RPS6KA1gi: 4506732 Hs.149957 polypeptide 1 1 26.7 −0.61 0.015 hypotheticalprotein FLJ20477 FLJ20477 gi: 8923441 Hs.259605 1 26.7 −0.84 0.007hypothetical protein FLJ20477 FLJ20477 gi: 1799134 Hs.259605 1 26.8−2.26 0.032 hypothetical protein FLJ12455 FLJ12455 gi: 11545792 Hs.109031 28.5 −0.75 <.005 tRNA selenocysteine associated protein SECP43 gi:8923459 Hs.266935 1 28.6 −0.87 <.005 TAF12 RNA polymerase II, TATA boxbind- TAF12 gi: 1345403 Hs.421646 ing protein (TBP)-associated factor,20 kDa 1 28.7 −0.63 0.016 glucocorticoid modulatory element GMEB1 gi:13435376 Hs.4069 binding protein 1 1 28.7 −0.78 0.005high-glucose-regulated protein 8 HGRG8 gi: 7705410 Hs.20993 1 29.1 −0.470.032 splicing factor, arginine/serine-rich 4 SFRS4 gi: 5032088 Hs.761221 29.2 −0.57 0.008 nuclear receptor binding factor 1 CGI-63 gi: 7705776Hs.183646 1 31.1 −0.49 0.037 hypothetical protein FLJ12650 FLJ12650 gi:13375663 Hs.436090 1 32.7 −0.67 0.006 KIAA1522 protein KIAA1522 gi:6588393 Hs.322735 1 32.9 −0.63 0.017 hypothetical protein FLJ90005FLJ90005 gi: 6729581 Hs.511807 1 32.9 −0.27 <.005 Homo sapienstranscribed sequence with gi: 6664283 Hs.416117 moderate similarity toprotein ref: NP_060265.1 (H. sapiens) hypothetical protein FLJ20378[Homo sapiens] 1 33.3 −0.96 <.005 polyhomeotic-like 2 (Drosophila) PHC2gi: 4758241 Hs.165263 3 14.1 −0.86 <.005 hypothetical protein MGC3222MGC3222 gi: 1384812 Hs.130330 3 14.1 −1.30 <.005 xeroderma pigmentosum,complementation XPC gi: 475156 Hs.320 group C 3 14.2 −0.47 0.033 LSM3homolog, U6 small nuclear RNA LSM3 gi: 7657314 Hs.111632 associated (S.cerevisiae) 4 153.2 −0.42 0.023 PET112-like (yeast) PET112L gi: 4758893Hs.119316 4 153.8 −0.38 0.043 F-box and WD-40 domain protein 7 FBXW7 gi:8922851 Hs.312503 (archipelago homolog, Drosophila) 4 174.8 −0.42 0.037UDP-N-acetyl-alpha-D-galactosamine:poly- GALNT7 gi: 8393408 Hs.156856peptide N-acetylgalactosaminyltransferase 7 (GalNAc-T7) 4 175 −0.420.027 Homo sapiens clone FLB9413 PRO2532 gi: 11493455 Hs.383372 mRNA,complete cds 4 175.1 −0.40 0.021 heart and neural crest derivativesexpressed 2 HAND2 gi: 12545383 Hs.388245 4 184.5 −0.73 <.005 dCMPdeaminase DCTD gi: 4740472 Hs.76894 4 185 −0.70 0.006collaborates/cooperates with ARF CARF gi: 8923039 Hs.32922 (alternatereading frame) protein 4 186 −0.74 <.005 interferon regulatory factor 2IRF2 gi: 4755144 Hs.83795 4 186.9 −0.38 0.05 hypothetical proteinDKFZp761O0113 DKFZp761O0113 gi: 8922176 Hs.42768 4 187 −0.40 0.028hypothetical protein FLJ11200 FLJ11200 gi: 8922937 Hs.368022 5 128.5−2.23 0.042 CGI-111 protein CGI-111 gi: 7705613 Hs.11085 6 106.7 −0.63<.005 APG5 autophagy 5-like (S. cerevisiae) APG5L gi: 7023451 Hs.11171 6107.1 −0.45 0.015 glutaminyl-tRNA synthase (glutamine- QRSL1 gi:12052881 Hs.406917 hydrolyzing)-like 1 6 107.6 −0.61 <.005 chromosome 6open reading frame 210 C6orf210 gi: 9966852 Hs.268733 6 108.2 −0.95<.005 SEC63-like (S. cerevisiae) SEC63 gi: 5393231 Hs.330767 6 108.5−0.48 0.01 sorting nexin 3 SNX3 gi: 6010168 Hs.12102 6 108.6 −0.53 0.007sorting nexin 3 SNX3 gi: 11765691 Hs.12102 6 109 −0.24 0.014 gi: 38210186 109.7 −0.75 <.005 CD164 antigen, sialomucin CD164 gi: 11943350Hs.43910 6 109.8 −0.77 <.005 sphingomyelin phosphodiesterase 2, neutralSMPD2 gi: 5101461 Hs.55235 membrane (neutral sphingomyelinase) 6 109.8−0.76 <.005 NEDD9 interacting protein with calponin NICAL gi: 12232438Hs.33476 homology and LIM domains 6 109.8 −0.54 0.008 zinc fingerprotein 450 ZNF450 gi: 7662127 Hs.409876 6 110.1 −0.36 0.04 KIAA0274KIAA0274 gi: 7662033 Hs.419998 6 110.5 −0.52 <.005 WAS protein family,member 1 WASF1 gi: 4507912 Hs.75850 6 110.5 −0.94 <.005 cell divisioncycle 40 homolog (yeast) CDC40 gi: 7706656 Hs.116674 6 110.9 −1.03 <.005cyclin-dependent kinase (CDC2-like) 11 CDK11 gi: 5100783 Hs.129836 6 111−1.36 <.005 cyclin-dependent kinase (CDC2-like) 11 CDK11 gi: 5689392Hs.129836 6 111.2 −0.41 <.005 adenosylmethionine decarboxylase 1 AMD1gi: 178517 Hs.159118 6 111.7 −0.76 <.005 REV3-like, catalytic subunit ofDNA REV3L gi: 4506482 Hs.232021 polymerase zeta (yeast) 6 114.3 −0.72<.005 histone deacetylase 2 HDAC2 gi: 4557640 Hs.3352 6 116.6 −0.84<.005 TSPY-like TSPYL gi: 12052783 Hs.458358 6 119.2 −0.10 <.005 ASF1anti-silencing function 1 homolog ASF1A gi: 7661591 Hs.292316 A (S.cerevisiae) 6 135.3 −0.65 0.019 HBS1-like (S. cerevisiae) HBS1L gi:6703779 Hs.221040 6 135.6 −0.57 0.016 Abelson helper integration stieAHI1 gi: 8923074 Hs.273294 6 136.9 −0.29 0.024 mitogen-activated proteinkinase kinase MAP3K5 gi: 1805499 Hs.151988 kinase 5 6 137.3 −0.58 <.005interleukin 20 receptor, alpha IL20RA gi: 7657690 Hs.288240 6 138.7−0.52 0.012 heme binding protein 2 HEBP2 gi: 7657602 Hs.439081 6 139−0.74 <.005 chromosome 6 open reading frame 80 C6orf80 gi: 12653928Hs.44468 6 139.4 −0.45 0.024 headcase homolog (Drosophila) HECA gi:7706434 Hs.6679 6 146.5 −0.76 <.005 glutamate receptor, metabotropic 1GRM1 gi: 6006005 Hs.32945 6 169.8 −0.25 0.047 PHD finger protein 10PHF10 gi: 11085906 Hs.435933 6 169.8 −0.61 0.011 PHD finger protein 10PHF10 gi: 8922799 Hs.435933 8 18 −0.44 0.026 N-acetyltransferase 1(arylamine N- NAT1 gi: 4505334 Hs.458430 acetyltransferase) 8 18.3 −0.65<.005 hypothetical protein DKFZp761K1423 DKFZp761K1423 gi: 8922171Hs.236438 8 18.5 −0.68 <.005 ADP-ribosylation factor guanine EFA6R gi:6085952 Hs.408177 nucleotide factor 6 8 20 −0.75 <.005 ATPase, H+transporting, lysosomal ATP6V1B2 gi: 4502310 Hs.295917 56/58 kDA, V1subunit B, isoform 2 8 28.6 −0.40 0.042 exostoses (multiple)-like 3EXTL3 gi: 2897904 Hs.9018 8 28.6 −0.49 0.02 exostoses (multiple)-like3:exostoses EXTL3 gi: 13623512 Hs.9018; (multiple)-like 3 Hs.9018 8 28.7−0.80 <.005 hypothetical protein FLJ10871 FLJ10871 gi: 8922725 Hs.155628 29.9 −0.99 <.005 hypothetical protein MGC8721 MGC8721 gi: 7706384Hs.279921 8 30 −0.96 <.005 leptin receptor overlapping transcript-like 1LEPROTL1 gi: 7662509 Hs.146585 8 30 −0.80 <.005 dynactin 6 DCTN6 gi:5730115 Hs.158427 8 30.3 −0.22 0.034 RNA binding protein with multiplesplicing RBPMS gi: 5803140 Hs.195825 8 30.5 −0.56 <.005 generaltranscription factor IIE, GTF2E2 gi: 4504194 Hs.77100 polypeptide 2,beta 34 kDA 8 30.6 −0.84 <.005 glutathione reductase GSR gi: 10835188Hs.414334 8 30.6 −0.42 0.022 reproduction 8 D8S2298E gi: 1913786Hs.153678 8 30.7 −0.46 <.005 protein phosphatase 2 (formerly 2A), PPP2CBgi: 4758951 Hs.80350 catalytic subunit, beta isoform 8 32.6 −0.08 0.039neuregulin 1 NRG1 gi: 7669513 Hs.172816 8 33.4 −1.04 <.005 RNA bindingprotein; RNA binding protein LOC84549 gi: 13625185 Hs.77135; Hs.77135 833.4 −0.53 <.005 hypothetical protein FLJ23263 FLJ23263 gi: 13376690Hs.288716 8 37.6 −0.75 <.005 chromosome 8 open reading frame 2; C8orf2gi: 10241715 Hs.125849; chromosome 8 open reading frame 2 Hs.125849 837.6 −1.00 <.005 proline synthetase co-transcribed PROSC gi: 6005841Hs.301959 homolog (bacterial) 8 131.3 −0.17 0.021 Homo sapiens cDNA:FLJ23601 fis, gi: 10440343 Hs.306918 clone LNG15501 9 2.1 −0.36 0.026SWI/SNF related, matrix associated, actin SMARCA2 gi: 6133361 Hs.396404dependent regulator of chromatin, subfamily a, member 2 9 2.6 −0.470.013 very low density lipoprotein receptor VLDLR gi: 437386 Hs.370422 92.8 −1.06 <.005 minor histocompatibility antigen HA-8 XTP5 gi: 7661865Hs.443866 9 5.3 −0.77 <.005 chromosome 9 open reading frame 46 C9orf46gi: 8923931 Hs.416649 9 5.5 −0.51 0.007 programmed cell death 1 ligand 2PDCD1LG2 gi: 13376849 Hs.61929 9 6 −1.15 <.005 RAN binding protein 6RANBP6 gi: 3538999 Hs.167496 9 14 −0.20 <.005 nuclear factor I/B NFIBgi: 13410807 Hs.302690 9 14.2 −0.50 <.005 nuclear factor I/B NFIB gi:4988418 Hs.302690 9 15.4 −0.56 <.005 small nuclear RNA activatingcomplex, SNAPC3 gi: 4507104 Hs.380092 polypeptide 3, 50 kDa 9 15.4 −0.65<.005 PC4 and SFRS1 interacting protein 2 PSIP2 gi: 3283351 Hs.351305 919 −0.94 <.005 Ras-related GTP binding A RRAGA gi: 5729998 Hs.432330 919 −0.43 0.026 hypothetical protein FLJ20060 FLJ20060 gi: 8923062Hs.54617 9 19.3 −0.91 <.005 ribosomal protein S6 RPS6 gi: 4506730Hs.408073 9 20.8 −1.14 <.005 KIAA1797 KIAA1797 gi: 8923357 Hs.257696 921.3 −1.03 <.005 KIAA1354 protein KIAA1354 gi: 2185814 Hs.147717 9 21.90.00 0.015 cyclin-dependent kinase inhibitor 2A CDKN2A gi: 4502748Hs.421349 (melanoma, p16, inhibits CDK4) 9 22 −0.15 <.005methylthioadenosine phosphorylase MTAP gi: 4378719 Hs.459541 12 42.4−1.31 <.005 PTK9 protein tyrosine kinase 9; PTK9 PTK9 gi: 4506274Hs.189075; protein tyrosine kinase 9 Hs.189075 12 43.8 −1.03 0.015putative glycolipid transfer protein LOC51054 gi: 7705683 Hs.334649 1244.6 −0.94 0.006 splicing factor, arginine/serine-rich 2, SFRS2IP gi:4759171 Hs.210367 interacting protein 12 46.4 −1.06 0.047 hypotheticalprotein FLJ20489 FLJ20489 gi: 8923451 Hs.438867 12 46.5 −2.40 0.043vitamin D (1,25-dihydroxyvitamin D3) VDR gi: 2824068 Hs.2062 receptor 1247.3 −0.54 0.036 hypothetical protein FLJ20436 FLJ20436 gi: 10434303Hs.268189 12 47.5 −0.89 0.007 calcium channel, voltage-dependent, betaCACNB3 gi: 463890 Hs.250712 3 subunit 12 47.5 −0.82 0.007 calciumchannel, voltage-dependent, beta CACNB3 gi: 463890 Hs.250712 3 subunit12 47.5 −0.58 0.028 DEAD (Asp-Glu-Ala-Asp) box polypeptide 23 DDX23 gi:2655201 Hs.130098 12 47.6 −0.72 0.023 FK506 binding protein 11, 19 kDaFKBP11 gi: 7706130 Hs.438695 12 47.6 −0.61 0.019 ADP-ribosylation factor3 ARF3 gi: 4502202 Hs.119177 12 47.6 −1.26 <.005 ADP-ribosylation factor3; ADP- ARF3 gi: 178980 Hs.119177; ribosylation factor 3 Hs.119177 1247.6 −0.48 0.045 protein kinase, AMP-activated, gamma 1 PRKAG1 gi:4506060 Hs.3136 non-catalytic subunit 12 47.7 −0.58 0.028lipocalin-interacting membrane receptor LIMR gi: 8922462 Hs.272838 1248.1 −1.12 <.005 spermatogenesis associated, serine-rich 2 SPATS2 gi:12751480 Hs.152982 12 48.2 −1.25 <.005 microspherule protein 1 MCRS1 gi:5453693 Hs.25313 12 48.4 −1.09 <.005 testis enhanced gene transcript(BAX TEGT gi: 2645728 Hs.35052 inhibitor 1) 12 48.6 −0.55 0.03 RacGTPase activating protein 1 RACGAP1 gi: 11015369 Hs.23900 12 49.6 −1.330.034 solute carrier family 11 (proton-coupled SLC11A2 gi: 11015990Hs.57435 divalent metal ion transporters), member 2 12 49.8 −2.85 0.034transcription factor CP2 TFCP2 gi: 5032174 Hs.154970 12 49.9 −1.32 <.005DAZ associated protein 2 DAZAP2 gi: 7661885 Hs.369761 12 49.9 −0.660.032 hypothetical protein from clone 643 LOC57228 gi: 13097236Hs.206501 12 50.6 −0.44 0.043 activin A receptor, type IB ACVR1B gi:12652986 Hs.371974 12 50.6 −0.58 0.035 activin A receptor, type IBACVR1B gi: 5912233 Hs.371974 12 50.7 −0.57 0.035 Homo sapiens cDNA clonegi: 7152120 Hs.444433 IMAGE: 5590288, partial cds 12 51.7 −1.36 0.012eukaryotic translation initiation factor 4B EIF4B gi: 4503532 Hs.9337912 51.8 −1.11 0.009 retinoic acid receptor, gamma RARG gi: 307424Hs.1497 12 51.9 −0.78 0.04 hypothetical protein MGC11308 MGC11308 gi:11975558 Hs.19210 12 51.9 −1.36 <.005 prefoldin 5 PFDN5 gi: 4505742Hs.288856 12 51.9 −0.76 0.044 chromosome 12 open reading frame 10C12orf10 gi: 11056017 Hs.400801 12 52.6 −0.56 0.042 homeo box C11 HOXC11gi: 7657165 Hs.127562 12 52.7 −0.73 0.01 homeo box C6 HOXC6 gi: 6709275Hs.820 12 52.8 −1.85 <.005 single-strand selective monofunctional SMUG1gi: 7657596 Hs.5212 uracil DNA glycosylase 12 52.9 −1.16 0.012 Homosapiens, clone IMAGE: 5288883, gi: 13284730 Hs.349283 mRNA 12 52.9 −1.600.034 heterogeneous nuclear ribonucleoprotein A1 HNRPA1 gi: 4504444Hs.356721 12 53 −4.54 0.034 costomer protein complex, subunit zeta 1COPZ1 gi: 7706336 Hs.181271 12 54.3 −1.01 <.005 GCN5 general control ofamino-acid GCN5L1 gi: 4503954 Hs.94672 synthesis 5-like 1 (yeast) 1254.4 −2.59 <.005 CD63 antigen (melanoma 1 antigen) CD63 gi: 4502678Hs.445570 12 54.4 −1.52 0.013 ORM1-like 2 (S. cerevisiae) ORMDL2 gi:7661819 Hs.13144 12 63.1 −0.90 0.005 exportin, tRNA (nuclear exportreceptor XPOT gi: 5811224 Hs.85951 for tRNAs) 12 63.1 −0.76 0.014TANK-binding kinase 1 TBK1 gi: 7019546 Hs.432466 12 63.3 −1.23 <.005glucosamine (N-acetyl)-6-sulfatase GNS gi: 10329021 Hs.334534(Sanfilippo disease IIID) 12 63.4 −0.78 0.013 glucosamine(N-acetyl)-6-sulfatase GNS gi: 4504060 Hs.334534 (Sanfilippo diseaseIIID) 12 63.8 −1.13 0.006 integral inner nuclear membrane protein MAN1gi: 7706606 Hs.105234 12 64.8 −0.71 0.012 CGI-119 protein CGI-119 gi:7706334 Hs.126372 12 65.9 −0.73 0.005 TBP-interacting protein TIP120Agi: 8924259 Hs.512638 12 65.9 −0.79 <.005 TBP-interacting proteinTIP120A gi: 4240146 Hs.512638 12 66.3 −0.70 0.006 dual-specificitytyrosine-(Y)- DYRK2 gi: 1666065 Hs.173135 phosphorylation regulatedkinase 2 12 67.3 −0.97 <.005 RAP1B, member of RAS oncogene family RAP1Bgi: 7661677 Hs.374418 12 67.4 −0.52 0.031 solute carrier family 35,member E2 SLC35E3 gi: 8922084 Hs.445043 12 68 −0.80 0.005glioma-amplified sequence-41 GAS41 gi: 5729837 Hs.4029 12 68.2 −0.530.05 chaperonin containing TCP1, subunit 2 (beta) CCT2 gi: 5453602 Hs.189772 12 78.7 −0.98 <.005 protein phosphatase 1, regulatory PPP1R12Agi: 5436140 Hs.377908 (inhibitor) subunit 12A 12 81.2 −0.81 <.005HSPC128 protein HSPC128 gi: 7661789 Hs.90527 12 86.9 −0.86 <.005hypothetical protein DKFZp434N2030 DKFZp434N2030 gi: 6708922 Hs.49420412 87 −0.52 0.045 hypothetical protein FLJ13615 FLJ13615 gi: 12711597Hs.288715 12 87.4 −0.47 0.03 KIT ligand KITLG gi: 4505174 Hs.1048 1288.2 −0.45 0.027 dual specificity phosphatase 6 DUSP6 gi: 13111942Hs.298654 12 88.4 −0.88 0.006 ATPase, Ca++ transporting, plasma ATP2B1gi: 7247996 Hs.20952 membrane 1 12 88.5 −0.73 <.005 ATPase, Ca++transporting, plasma ATP2B1 gi: 184269 Hs.20952 membrane 1 12 91 −0.650.008 B-cell translocation gene 1, anti-proliferative BTG1 gi: 4502472Hs.255935 16 2.5 −0.60 0.026 ATPase, H+ transporting, lysosomal ATP6V0Cgi: 4502312 Hs.389107 16 kDa, V0 subunit c 16 2.5 −0.62 0.019 ATPase, H+transporting, lysosomal ATP6V0C gi: 189675 Hs.389107 16 kDa, V0 subunitc 16 2.5 −0.70 <.005 CGI-14 protein CGI-14 gi: 7705595 Hs.433499 16 2.5−0.14 0.02 3-phosphoinositide dependent protein PDPK1 gi: 4505694Hs.154729 kinase-1 16 2.5 −0.92 <.005 3-phosphoinositide dependentprotein PDPK1 gi: 2407612 Hs.154729 kinase-1 16 2.8 −0.15 <.005serine/arginine repetitive matrix 2 SRRM2 gi: 4739778 Hs.433343 16 2.8−0.61 0.01 serine/arginine repetitive matrix 2 SRRM2 gi: 4531907Hs.433343 17 45.4 −0.10 0.011 golgi SNAP receptor complex member 2 GOSR2gi: 12711466 Hs.432552 18 37.8 −0.70 <.005 phosphoinositide-3-kinase,class 3 PIK3C3 gi: 4505800 Hs.418150 18 41.9 −0.70 <.005 ATP synthase,H+ transporting, mitochondrial ATP5A1 gi: 4573764 Hs.298280 F1 complex,alpha subunit, isoform 1, cardiac muscle 18 42.6 −0.41 0.031Msx-interacting-zinc finger MIZ1 gi: 10720797 Hs.441069 18 42.6 −0.500.008 Msx-interacting-zinc finger MIZ1 gi: 3643114 Hs.441069 18 42.9−0.60 0.006 HSPC039 protein HSPC039 gi: 7770186 Hs.406542 18 66.1 −0.420.034 suppressor of cytokine signaling 4 SOCS4 gi: 4757991 Hs.44439 1872.8 −0.48 0.011 myelin basic protein MBP gi: 4505122 Hs.408543 18 75.3−0.31 0.005 nuclear factor of activated T-cells, NFATC1 gi: 500631Hs.512591 cytoplasmic, calcineurin-dependent 1 38 75.5 −0.60 <.005 CTD(carboxy-terminal domain, RNA CTDP1 gi: 4758093 Hs.4076 polymerase II;polypeptide A) phosphatase, subunit 1 18 75.7 −0.58 <.005 hypotheticalprotein FLJ22378 FLJ22378 gi: 13376629 Hs.288284 18 75.8 −0.61 <.005similar to S. pombe dim1+ DIM1 gi: 12654440 Hs.433683 18 75.8 −0.53<.005 hypothetical protein FLJ21172 FLJ21172 gi: 13376184 Hs.444642 1875.9 −0.28 <.005 KIAA0863 protein KIAA0863 gi: 10434228 Hs.131915 1951.5 −0.81 0.018 protein phosphatase 5, catalytic subunit PPP5C gi:5453957 Hs.431861 19 59.3 −0.32 <.005 PRP31 pre-mRNA processing factor31 PRPF31 gi: 7661653 Hs.312927 homolog (yeast) 19 59.9 −0.40 0.047killer cell immunoglobulin-like receptor, KIR2DL1 gi: 897908 Hs.512572two domains, long cytoplasmic tail, 1 19 60.1 −0.18 0.016 naturalcytotoxicity triggering receptor 1 NCR1 gi: 4758691 Hs.97084 21 45 −0.510.025 SMT3 suppressor of mif two 3 homolog 1 SMT3H1 gi: 5902095 Hs.85119(yeast) 21 45.1 −0.84 <.005 pituitary tumor-transforming 1 interactingPTTG1IP gi: 11038670 Hs.369026 protein 21 46.9 −0.31 0.05 HMT1 hnRNPmethyltransferase-like 1 HRMT1L1 gi: 4504494 Hs.154163 (S. cerevisiae)22 21.3 −0.42 0.038 POM121 membrane glycoprotein-like 1 (rat) POM121L1gi: 7657468 Hs.380370 22 21.3 −0.57 0.012 immunoglobulin lambda joining3 IGLJ3 gi: 13171335 Hs.102950 22 21.8 −0.50 0.015 RAB36, member RASoncogene family RAB36 gi: 6049163 Hs.369557 22 21.9 −0.64 <.005breakpoint cluster region BCR gi: 11038638 Hs.446394 22 22.2 −0.65 0.009immunoglobulin lambda-like polypeptide 1 IGLL1 gi: 13399297 Hs.348935 2222.3 −0.52 0.013 Homo sapiens, clone IMAGE: 5728597, gi: 292400Hs.272302 mRNA 22 22.4 −0.45 0.03 matrix metalloproteinase 11(stromelysin 3) MMP11 gi: 5177469 Hs.143751 22 22.4 −1.28 <.005 SWI/SNFrelated, matrix associated, actin SMARCB1 gi: 4507076 Hs.512700dependent regulator of chromatin, subfamily b, member 1 22 22.7 −1.44<.005 glutathione S-transferase theta 1 GSTT1 gi: 4504184 Hs.268573 2223.2 −0.58 0.028 small nuclear ribonucleoprotein D3 SNRPD3 gi: 4759159Hs.356549 22 25.1 −0.48 0.014 Hermansky-Pudlak syndrome 4 HPS4 gi:5420802 Hs.441481 22 25.1 −0.64 <.005 Hermansky-Pudlak syndrome 4 HPS4gi: 11559920 Hs.441481 22 25.2 −0.15 <.005 tuftelin interacting protein11 TFIP11 gi: 5262598 Hs.20225 22 25.3 −0.52 0.046 crystallin, beta A4CRYBA4 gi: 4503058 Hs.57690 22 26.4 −0.67 0.033 meningiorna (disruptedin balanced MN1 gi: 4505222 Hs.268515 translocation) 1 22 27.4 −0.730.035 CHK2 checkpoint homolog (S. pombe) CHEK2 gi: 13278893 Hs.146329 2228.2 −0.07 0.033 chromosome 22 open reading frame 19 C22orf19 gi:13177658 Hs.75361 22 28.5 −0.55 0.024 ASC-1 complex subunit P100ASC1p100 gi: 5419897 Hs.436407 22 29 −0.84 <.005 splicing factor 3a,subunit 1, 120 kDa SF3A1 gi: 5032086 Hs.406277 22 29.1 −0.87 <.005SEC14-like 2 (S. cerevisiae) SEC14L2 gi: 7110714 Hs.430576 22 30 −1.48<.005 zinc finger protein 278 ZNF278 gi: 9954374 Hs.27801 22 30.2 −0.73<.005 KIAA0542 gene product KIAA0542 gi: 6635200 Hs.62209 22 30.2 −1.14<.005 KIAA0542 gene product KIAA0542 gi: 3043607 Hs.62209 22 30.3 −0.66<.005 phosphatidylserine decarboxylase PISD gi: 13489111 Hs.8128 22 30.3−0.84 <.005 KIAA0542 gene product KIAA0542 gi: 5596770 Hs.62209 22 30.6−0.72 <.005 KIAA0645 gene product KIAA0645 gi: 7662221 Hs.435022 22 30.6−0.45 0.021 tyrosine 3-monoxygenase/tryptophan 5-mono- YWHAH gi: 4507950Hs.226755 xygenase activation protein, eta polypeptide 22 31.2 −0.720.011 Homo sapiens, clone IMAGE: 4818531, gi: 10030150 Hs.150167 mRNA 2231.5 −0.50 0.027 tissue inhibitor of metalloproteinase 3 TIMP3 gi:1519557 Hs.245188 (Sorsby fundus dystrophy, pseudoinflammatory) 22 34−0.37 0.047 target of myb1 (chicken) TOM1 gi: 4885636 Hs.9482 22 34.3−0.48 0.039 apolipoprotein L, 6 APOL6 gi: 13449280 Hs.257352 22 34.4−0.76 <.005 RNA binding motif protein 9 RBM9 gi: 1267308 Hs.433574 2234.8 −0.62 0.013 apolipoprotein L, 3 APOL3 gi: 7656972 Hs.241535 22 34.9−0.66 <.005 apolipoprotein L, 2 APOL2 gi: 13325155 Hs.398037 22 34.9−0.44 0.032 myosin, heavy polypeptide 9, non-muscle MYH9 gi: 5448699Hs.146550 22 35 −0.84 <.005 thioredoxin 2 TXN2 gi: 4200326 Hs.211929 2235.1 −1.23 <.005 thioredoxin 2 TXN2 gi: 9280552 Hs.211929 22 35.1 −1.11<.005 eukaryotic translation initiation factor 3, EIF3S7 gi: 4503522Hs.55682 subunit 7 zeta, 66/67 kDa 22 35.6 −0.37 0.046 thiosulfatesulfurtransferase (rhodanese) TST gi: 1877030 Hs.351863 22 35.6 −0.640.005 mercaptopyruvate sulfurtransferase MPST gi: 13489090 Hs.248267 2235.6 −0.58 0.01 hypothetical protein FLJ12242 FLJ12242 gi: 13489098Hs.94810 22 36.1 −0.53 0.033 manic fringe homolog (Drosophila) MFNG gi:5175720 Hs.371768 22 36.1 −0.20 0.015 caspase recruitment domain family,CARD10 gi: 5877877 Hs.57973 member 10 22 36.2 −1.08 <005 golgiassociated, gamma adaptin ear GGA1 gi: 9558728 Hs.405689 containing, ARFbinding protein 1 22 36.2 −0.62 0.023 golgi associated, gamma adaptinear GGA1 gi: 5858473 Hs.405689 containing, ARF binding protein 1 22 36.2−0.61 0.026 SH3-domain binding protein 1 SH3BP1 gi: 11545732 Hs.51195422 36.4 −0.76 0.021 glycine C-acetyltransferase (2-amion-3- GCAT gi:7657117 Hs.54609 ketobutyrate coenzyme A ligase) 22 36.5 −1.41 >.005polymerase (RNA) II (DNA directed) POLR2F gi: 1309770 Hs.46405polypeptide F 22 36.9 −1.89 0.042 casein kinase 1, epsilon CSNK1E gi:6471575 Hs.355669 22 37.1 −0.88 <.005 KDEL (Lys-Asp-Glu-Leu) endoplasmicKDELR3 gi: 8051612 Hs.250696 reticulum protein retention receptor 3 2237.1 −0.54 0.026 DMC1 dosage suppressor of mck1 homolog DMC1 gi: 106600Hs.339396 meiosis-specific homologens recombination (yeast) 22 37.3−0.48 0.028 chromosome 22 open reading frame 2 C22orf2 gi: 7656941Hs.334911 22 37.3 −0.47 0.038 transiocase of outer mitochondrial TOMM22gi: 9910381 Hs.285005 membrane 22 homolog (yeast) 22 37.3 −0.75 <.005KIAA0063 gene product KIAA0063 gi: 7661887 Hs.3094 22 37.3 −0.41 0.036unc-84 homolog B (C. elegans) UNC84B gi: 4582132 Hs.406612 22 37.3 −0.500.016 GTP binding protein 1 GTPBP1 gi: 1916924 Hs.283677 22 37.3 −1.04<.005 GTP binding protein 1 GTPBP1 gi: 7661735 Hs.283677 22 37.8 −0.430.008 platelet-derived growth factor beta PDGFB gi: 11012269 Hs.1976polypeptide (sitnian sarcoma viral (v-sis) oncogene homolog) 22 38 −0.540.042 mitogen-activated protein kinase kinase MAP3K7IP1 gi: 5174702Hs.403927 kinase 7 interacting protein 1 22 38.1 −0.56 0.047 mannosyl(beta-1,4-)-glycoprotein beta- MGAT3 gi: 6031184 Hs.2768081,4-N-acetylglucosaminyltransferase 22 38.1 −0.64 0.03 hypotheticalprotein FLJ20232 FLJ20232 gi: 12803520 Hs.505742 22 38.1 −0.62 0.018hypothetical protein FLJ20232 FLJ20232 gi: 1524716 Hs.505742 22 38.1−0.48 0.041 activating transcription factor 4 (tax- ATF4 gi: 4502264Hs.181243 responsive enhancer element B67) 22 38.2 −0.52 0.043 mannosyl(beta-1,4-)-glycoprotein beta- MGAT3 gi: 4914501 Hs.2768081,4-N-acetylglucosaminyltransferase 22 39 −0.26 0.022 hypotheticalprotein DJ1042K10.2 DJ1042K10.2 gi: 11034850 Hs.22129 Ref Seq SEQ IDNO.: Ref Seq Prot SEQ ID NO.: Chromosome Locus Link Regulation ProbesmRna ID Nuc. ID A.A. 1 249 DOWN 215783_s_at NM_000478 427 NP_000469 11781 23028 DOWN 212348_s_at NM_015013 428 NP_055828 1179 1 1870 DOWN207042_at NM_004091 429 NP_004082 1180 1 11313 DOWN 202292_x_atNM_007260 430 NP_009191 1181 1 2582 DOWN 202528_at NM_000403 431NP_000394 1182 1 3155 DOWN 202772_at NM_000191 432 NP_000182 1183 111313 DOWN 215568_x_at NM_007260 433 NP_009191 1184 1 2517 DOWN202838_at NM_000147 434 NP_000138 1185 1 10250 DOWN 201225_s_atNM_005839 435 NP_005830 1186 1 25932 DOWN 201559_s_at NM_013943 436NP_039234 1187 1 57035 DOWN 209006_s_at NM_020317 437 NP_064713 1188 13925 DOWN 200783_s_at NM_005563 438 NP_005554 1189 1 51042 DOWN204175_at NM_015871 439 NP_056955 1190 1 3151 DOWN 208668_x_at NM_005517440 NP_005508; 1191 NP_116138 1 6195 DOWN 203379_at NM_002953 441NP_002944 1192 1 55650 DOWN 219238_at NM_017837 442 NP_060307 1193 155650 DOWN 51146_at NM_017837 443 NP_060307 1194 1 63906 DOWN 218895_atNM_022078 444 NP_071361 1195 1 54952 DOWN 218977_s_at NM_017846 446NP_060316 1197 1 6883 DOWN 209463_s_at NM_005644 447 NP_005635 1198 110691 DOWN 220938_s_at NM_006582; 448; 449 NP_006573; 1199; 1200NM_024482 NP_077808 1 51441 DOWN 217812_at NM_016258 450 NP_057342 12011 6429 DOWN 201696_at NM_005626 451 NP_005617 1202 1 51102 DOWN218664_at NM_016011 452 NP_057095 1203 1 79570 DOWN 219438_at NM_024522453 NP_078798 1204 1 57648 DOWN 212048_s_at XM_036299 454 XP_036299 12051 127544 DOWN 213038_at NM_153341 455 NP_699172 1206 1 DOWN 212172_at1501  — 1 1912 DOWN 200919_at NM_004427; 456; 457 NP_004418; 1207; 1208NM_198040 NP_932157 3 79188 DOWN 217795_s_at NM_024334 459 NP_0773101210 3 7508 DOWN 209375_at NM_004628 460 NP_004619 1211 3 27258 DOWN202209_at NM_014463 461 NP_055278 1212 4 5188 DOWN 204300_at NM_004564462 NP_004555 1213 4 55294 DOWN 218751_s_at NM_018315; 463; 464NP_060785; 1214; 1215 NM_033632 NP_361014 4 51809 DOWN 218313_s_atNM_017423 465 NP_059119 1216 4 DOWN 210918_at 1502  1503 4 9464 DOWN220480_at NM_021973 466 NP_068808 1217 4 1635 DOWN 201571_s_at NM_001921467 NP_001912 1218 4 55602 DOWN 218929_at NM_017632 468 NP_060102 1219 43660 DOWN 203275_at NM_002199 469 NP_002190 1220 4 55805 DOWN207797_s_at NM_018409 470 NP_060879 1221 4 55325 DOWN 218449_atNM_018359 471 NP_060829 1222 5 51015 DOWN 218170_at NM_016048 472NP_057132 1223 6 9474 DOWN 202511_s_at NM_004849 473 NP_004840 1224 655278 DOWN 218948_at NM_018292 474 NP_060762 1225 6 57107 DOWN 219307_atNM_020381 475 NP_065114 1226 6 11231 DOWN 201915_at NM_007214 476NP_009145 1227 6 8724 DOWN 200067_x_at NM_003795; 477; 478; 479NP_003786; 1228; 1229; 1230 NM_152827; NP_690040; NM_152828 NP_690041 68724 DOWN 213545_x_at NM_003795; 480; 481; 482 NP_003786; 1231; 1232;1233 NM_152827; NP_690040; NM_152828 NP_690041 6 DOWN 217185_s_at — — 68763 DOWN 208654_s_at NM_006016 483 NP_006007 1234 6 6610 DOWN 214206_atNM_003080 484 NP_003071 1235 6 64780 DOWN 218376_s_at NM_022765 485NP_073602 1236 6 9841 DOWN 205340_at XM_376525 486 XP_376525 1237 6 9896DOWN 203656_at NM_014845 487 NP_055660 1238 6 8936 DOWN 204165_atNM_003931 488 NP_003922 1239 6 51362 DOWN 203377_s_at NM_015891 489NP_056975 1240 6 23097 DOWN 212897_at NM_015076 490 NP_055891 1241 623097 DOWN 212899_at NM_015076 491 NP_055891 1242 6 262 DOWN 201196_s_atNM_001634 492 NP_001625 1243 6 5980 DOWN 208070_s_at NM_002912 493NP_002903 1244 6 3066 DOWN 201833_at NM_001527 494 NP_001518 1245 6 7259DOWN 221493_at XM_371844 495 XP_371844 1246 6 25842 DOWN 203427_atNM_014034 496 NP_054753 1247 6 10767 DOWN 209315_at NM_006620 497NP_006611 1248 6 54806 DOWN 220841_s_at NM_017651 498 NP_060121 1249 64217 DOWN 203836_s_at NM_005923 499 NP_005914 1250 6 53832 DOWN219115_s_at NM_014432 500 NP_055247 1251 6 23593 DOWN 203430_atNM_014320 501 NP_055135 1252 6 25901 DOWN 209479_at NM_015439 502NP_056254 1253 6 51696 DOWN 218603_at NM_016217 503 NP_057301 1254 62911 DOWN 207299_s_at NM_000838 504 NP_000829 1255 6 55274 DOWN221786_at NM_018288; 505; 506 NP_060758: 1256; 1257 NM_133325 NP_5798666 55274 DOWN 219126_at NM_018288; 507; 508 NP_060758; 1258; 1259NM_133325 NP_579866 8 9 DOWN 214440_at NM_000662 509 NP_000653 1260 855358 DOWN 218613_at NM_018422 510 NP_060892 1504 8 23362 DOWN203354_s_at NM_015310 511 NP_056125 1261 8 526 DOWM 201089_at NM_001693512 NP_001684 1262 8 2137 DOWN 209202_s_at NM_001440 513 NP_001431 12638 2137; 2137 DOWN 211051_s_at NM_001440 514 NP_001431 1264 8 55756 DOWN203941_at NM_018250 515 NP_060720 1265 8 51669 DOWN 200847_s_atNM_016127 516 NP_057211 1266 8 23484 DOWN 202594_at NM_015344 517NP_056159 1267 8 10671 DOWN 203261_at NM_006571 518 NP_006562 1268 811030 DOWN 207836_s_at NM_006867 519 NP_006858 1269 8 2961 DOWN202680_at NM_002095 520 NP_002086 1270 8 2936 DOWN 205770_at NM_000637521 NP_000628 1271 8 7993 DOWN 215983_s_at NM_005671 522 NP_005662 12728 5516 DOWN 201375_s_at NM_004156 523 NP_004147 1273 8 3084 DOWN206237_s_at NM_004495; 524; 525; 526; 527; NP_004486; 1274; 1275; 1276;NM_013956; 528; 529; 530; 531; NP_039250; 1277; 1278; 1279; NM_013957;532 NP_039251; 1280; 1281; 1282 NM_013958; NP_039252; NM_013959;NP_039253; NM_013960; NP_039254; NM_013961; NP_039255; NM_013962;NP_039256; NM_013964 NP_039258 8 84549; DOWN 211686_s_at NM_032509 533NP_115898 1283 84549 8 80185 DOWN 219124_at NM_025115 534 NP_079391 12848 11160; DOWN 221543_s_at NM_007175; 535; 536 NP_009106 1285 11160NM_007175 8 11212 DOWN 214545_s_at NM_007198 537 NP_009129 1286 8 DOWN216416_at 1505  — 9 6595 DOWN 212257_s_at NM_003070; 538; 539 NP_003061;1287; 1288 NM_139045 NP_620614 9 7436 DOWN 209822_s_at NM_003383 540NP_003374 1289 9 9933 DOWN 203712_at NM_014878 541 NP_055693 1290 955848 DOWN 218992_at NM_018465 542 NP_060935 1291 9 80380 DOWN220049_s_at NM_025239 543 NP_079515 1292 9 26953 DOWN 213019_atNM_012416 544 XP_039701 — 9 4781 DOWN 213029_at NM_005596 545 NP_0055871293 9 4781 DOWN 209289_at NM_005596 546 NP_005587 1294 9 6619 DOWN204001_at NM_003084 547 NP_003075 1295 9 11168 DOWN 209337_at NM_021144;548; 549 NP_066967; 1296; 1297 NM_033222 NP_150091 9 10670 DOWN201628_s_at NM_006570 550 NP_006561 1298 9 54801 DOWN 218602_s_atNM_017645 551 NP_060115 1299 9 6194 DOWN 201254_x_at NM_001010 552NP_001001 1300 9 54914 DOWN 218503_at NM_017794 553 NP_060264 1301 955958 DOWN 213233_s_at NM_018847 554 NP_061335 1302 9 1029 DOWN207039_at NM_000077; 556; 557; 558; NP_000068; 1304; 1305; 1306NM_058195; 559 NP_478102; NM_058196; NP_478103; NM_058197 NP_478104 94507 DOWN 211363_s_at NM_002451 560 NP_002442 1307 12 5756; 5756 DOWN201745_at NM_002822; 561; 562; 563; 564 NP_002813; 1308; 1309 NM_198974;NP_945325 NM_002822; NM_198974 12 51054 DOWN 220157_x_at NM_015899 565NP_056983 1310 12 9169 DOWN 206989_s_at NM_004719 566 NP_004710 1311 1255652 DOWN 218417_s_at NM_017842 567 NP_060312 1312 12 7421 DOWN204255_s_at NM_000376 568 NP_000367 1313 12 54934 DOWN 221821_s_atNM_017822 569 NP_060292 1314 12 784 DOWN 209530_at NM_000725 570NP_000716 1315 12 784 DOWN 34726_at NM_000725 571 NP_000716 1316 12 9416DOWN 40465_at NM_004818 572 NP_004809 1317 12 51303 DOWN 219117_s_atNM_016594 573 NP_057678 1318 12 377 DOWN 200011_s_at NM_001659 574NP_001650 1319 12 377; 377 DOWN 211622_s_at NM_001659 575 NP_001650 132012 5571 DOWN 201805_at NM_002733 576 NP_002724 1321 12 55716 DOWN220036_s_at NM_018113 577 NP_060583 1322 12 65244 DOWN 218324_s_atNM_023071 578 NP_075559 1323 12 10445 DOWN 202556_s_at NM_006337 579NP_006328 1324 12 7009 DOWN 200803_s_at NM_003217 580 NP_003208 1325 1229127 DOWN 222077_s_at NM_013277 581 NP_037409 1326 12 4891 DOWN203123_s_at NM_000617 582 NP_000608 1327 12 7024 DOWN 207627_s_atNM_005653 583 NP_005644 1328 12 9802 DOWN 200794_x_at NM_014764 584NP_055579 1329 12 57228 DOWN 209679_s_at NM_020467 585 NP_065200 1330 1291 DOWN 205209_at NM_004302; 587; 588; 589 NP_004293; 1332; 1333; 1334NM_020327; NP_064732; NM_020328 NP_064733 12 91 DOWN 213198_atNM_004302; 590; 591; 592 NP_004293; 1335; 1336; 1337 NM_020327;NP_064732; NM_020328 NP_064733 12 DOWN 222304_x_at 1506  — 12 1975 DOWN211937_at NM_001417 593 NP_001408 1338 12 5916 DOWN 217178_at NM_000966594 NP_000957 1339 12 84975 DOWN 212861_at NM_032889 595 NP_116278 134012 5204 DOWN 207132_x_at NM_002624; 596; 597; 598 NP_002615; 1341; 1342;1343 NM_145896; NP_665903; NM_145897 NP_665904 12 60314 DOWN 218220_atNM_021640 599 NP_067653 1344 12 3227 DOWN 206745_at NM_014212 600NP_055027 1345 12 3223 DOWN 206194_at NM_004503; 601; 602 NP_004494;1346; 1347 NM_153693 NP_710160 12 23583 DOWN 218685_s_at NM_014311 603NP_055126 1348 12 DOWN 212126_at 1507  — 12 3178 DOWN 200016_x_atNM_002136; 604; 605 NP_002127; 1349; 1350 NM_031157 NP_112420 12 22818DOWN 217726_at NM_016057 606 NP_057141 1351 12 2647 DOWN 202592_atNM_001487 607 NP_001478 1352 12 967 DOWN 200663_at NM_001780 608NP_001771 1353 12 29095 DOWN 218556_at NM_014182 609 NP_054901 1354 1211260 DOWN 212160_at NM_007235 614 NP_009166 1359 12 29110 DOWN218520_at NM_013254 615 NP_037386 1360 12 2799 DOWN 212334_at NM_002076616 NP_002067 1361 12 2799 DOWN 203676_at NM_002076 617 NP_002067 136212 23592 DOWN 218604_at NM_014319 618 NP_055134 1363 12 51643 DOWN219206_x_at NM_016056 619 NP_057140 1364 12 55832 DOWN 207483_s_atNM_018448 620 NP_060918 1365 12 55832 DOWN 208838_at NM_018448 621NP_060918 1366 12 8445 DOWN 202968_s_at NM_003583; 622; 623 NP_003574;1367; 1368 NM_006482 NP_006473 12 5908 DOWN 200833_s_at NM_015646 624NP_056461 1369 12 55508 DOWN 218988_at NM_018656 625 NP_061126 1370 128089 DOWN 218911_at NM_006530 626 NP_006521 1371 12 10576 DOWN201947_s_at NM_006431 627 NP_006422 1372 12 4659 DOWN 201603_atNM_002480 628 NP_002471 1373 12 29080 DOWN 218936_s_at NM_014167 629NP_054886 1374 12 91298 DOWN 213701_at 1508  — 12 80184 DOWN 221683_s_atNM_025114 630 NP_079390 1375 12 4254 DOWN 207029_at NM_000899; 631; 632NP_000890; 1376; 1377 NM_003994 NP_003985 12 1848 DOWN 208891_atNM_001946; 633; 634 NP_001937; 1378; 1379 NM_022652 NP_073143 12 490DOWN 212930_at NM_001682 635 NP_001673 1380 12 490 DOWN 209281_s_atNM_001682 636 NP_001673 1381 12 694 DOWN 200921_s_at NM_001731 637NP_001722 1382 16 527 DOWN 200954_at NM_001694 638 NP_001685 1383 16 527DOWN 36994_at NM_001694 639 NP_001685 1384 16 51005 DOWN 219082_atNM_015944 640 NP_057028 1385 16 5170 DOWN 204524_at NM_002613 641NP_002604 1386 16 5170 DOWN 32029_at NM_002613 642 NP_002604 1387 1623524 DOWN 208610_s_at NM_016333 643 NP_057417 1388 16 23524 DOWN213877_x_at NM_016333 644 NP_057417 1389 17 9570 DOWN 210009_s_atNM_004287; 233; 234 NP_004278; 984; 985 NM_054022 NP_473363 18 5289 DOWN204297_at NM_002647 645 NP_002638 1390 18 498 DOWN 213738_s_at NM_004046646 NP_004037 1391 18 9063 DOWN 214593_at NM_004671; 647; 648 NP_004662;1392; 1393 NM_173206 NP_775298 18 9063 DOWN 37433_at NM_004671; 649; 650NP_004662; 1394; 1395 NM_173206 NP_775298 18 51124 DOWN 211406_atNM_016097 651 NP_057181; 1396 NP_060992 18 9306 DOWN 214462_at NM_004232652 NP_004223 1397 18 4155 DOWN 207323_s_at NM_002385 653 NP_002376 139818 4772 DOWN 210161_at NM_006162; 654; 655; 666; 657; NP_006153; 1399;1400; 1401; NM_172387; 658 NP_765975; 1402; 1403 NM_172388; NP_765976;NM_172389; NP_765977; NM_172390 NP_765978 38 9150 DOWN 205035_atNM_004715; 659; 660 NP_004706; 1404; 1405 NM_048368 NP_430255 18 80148DOWN 218208_at NM_025078 661 NP_079354 1406 18 10907 DOWN 202835_atNM_006701 662 NP_006692 1407 18 79863 DOWN 219419_at NM_024805 663NP_079081 1408 18 22850 DOWN 203321_s_at XM_377498 664 XP_377498 1409 195536 DOWN 201979_s_at NM_006247 319 NP_006238 1070 19 26121 DOWN202408_s_at NM_015629 370 NP_056444 1121 19 3802 DOWN 210890_x_atNM_014218 665 NP_055033 1410 19 9437 DOWN 207860_at NM_004829 666NP_004820 1411 21 6612 DOWN 200740_s_at NM_006936 667 NP_008867 1412 21754 DOWN 200677_at NM_004339 668 NP_004330 1413 21 3275 DOWN 202098_s_atNM_001535 669 NP_001526 1414 22 25812 DOWN 214570_x_at NM_014348 670NP_055163 1415 22 28831 DOWN 216846_at 1509  1510 22 9609 DOWN211471_s_at NM_004914 671 NP_004905 1416 22 613 DOWN 202315_s_atNM_004327; 672; 673 NP_004318; 1417; 1418 NM_021574 NP_067585 22 3543DOWN 206660_at NM_020070; 674; 675 NP_064455; 1419; 1420 NM_152855NP_690594 22 375159 DOWN 215816_at 1511  1512 22 4320 DOWN 203876_s_atNM_005940 676 NP_005931 1421 22 6598 DOWN 206532_at NM_003073 677NP_003064 1422 22 2952 DOWN 203815_at NM_000853 678 NP_000844 1423 226634 DOWN 202567_at NM_004175 679 NP_004166 1424 22 89781 DOWN 54037_atNM_022081; 680; 681; 682; 683; NP_071364; 1425; 1426; 1427; NM_152840;684 NP_690053; 1428; 1429 NM_152841; NP_690054; NM_152842; NP_690055;NM_152843 NP_690056 22 89781 DOWN 218402_s_at NM_022081; 685; 686; 687;688; NP_071364; 1430; 1431; 1432; NM_152840; 689 NP_690053; 1433; 1434NM_152841; NP_690054; NM_152842; NP_690055; NM_152843 NP_690056 22 24144DOWN 202750_s_at NM_012143 690 NP_036275 1435 22 1413 DOWN 206843_atNM_001886 691 NP_001877 1436 22 4330 DOWN 205330_at NM_002430 692NP_002421 1437 22 11200 DOWN 210416_s_at NM_007194; 693; 694; NP_009125;1438; 1439 NM_145862 NP_665861 22 8563 DOWN 209418_s_at 1513  1514 2284164 DOWN 215684_s_at NM_032204 695 NP_115580 1440 22 10291 DOWN201357_s_at NM_005877 696 NP_005868 1441 22 23541 DOWN 204541_atNM_012429 697 NP_036561 1442 22 23598 DOWN 209431_s_at NM_014323; 698;699; 700; 701 NP_055138; 1443; 1444; 1445; NM_032050; NP_114439; 1446NM_032051; NP_114440; NM_032052 NP_114441 22 9814 DOWN 213431_x_atXM_038520 702 XP_038520 1447 22 9814 DOWN 36545_s_at XM_038520 703XP_038520 1448 22 23761 DOWN 202392_s_at NM_014338 704 NP_055153 1449 229814 DOWN 215699_x_at XM_038520 705 XP_038520 1450 22 9681 DOWN205223_at XM_377498 706 XP_376007 1451 22 7533 DOWN 201020_at NM_003405707 NP_003396 1452 22 DOWN 215762_at 1515  — 22 7078 DOWN 201149_s_atNM_000362 708 NP_000353 1453 22 10043 DOWN 202807_s_at NM_005488 709NP_005479 1454 22 80830 DOWN 219716_at NM_030641 710 NP_085144 1455 2223543 DOWN 212104_s_at NM_014309 711 NP_055124 1456 22 80833 DOWN221087_s_at NM_014349; 712; 713; 714; 715; NP_055164; 1457; 1458; 1459;NM_030644; 716; 717 NP_085147; 1460; 1461; 1462 NM_145639; NP_663614;NM_145640; NP_663615; NM_145641; NP_663616; NM_145642 NP_663617 22 23780DOWN 221653_x_at NM_030882; 718; 719 NP_112092; 1463; 1464 NM_145637NP_663612 22 4627 DOWN 211926_s_at NM_002473 720 NP_002464 1465 22 25828DOWN 209077_at NM_012473 721 NP_036605 1466 22 25828 DOWN 209078_s_atNM_012473 722 NP_036605 1467 22 8664 DOWN 200005_at NM_003753 723NP_003744 1468 22 7263 DOWN 209605_at NM_003312 724 NP_003303 1469 224357 DOWN 203524_s_at NM_021126 725 NP_066949 1470 22 79734 DOWN205561_at NM_024681 726 NP_078957 1471 22 4242 DOWN 213783_at NM_002405727 NP_002396 1472 22 29775 DOWN 214207_s_at NM_014550 728 NP_0553651473 22 26088 DOWN 218114_at NM_013365 729 NP_037497 1474 22 26088 DOWN45572_s_at NM_013365 730 NP_037497 1475 22 23616 DOWN 213633_atNM_018957 731 NP_061830 1476 22 23464 DOWN 205164_at NM_014291 732NP_055106 1477 22 5435 DOWN 209511_at NM_021974 733 NP_068809 1478 221454 DOWN 222015_at NM_001894; 734; 735 NP_001885; 1479; 1480 NM_152221NP_689407 22 11015 DOWN 204017_at NM_006855; 736; 737 NP_006846; 1481;1482 NM_016657 NP_057839 22 11144 DOWN 208382_s_at NM_007068 738NP_008999 1483 22 25776 DOWN 203450_at NM_015373 739 NP_056188 1484 2256993 DOWN 217960_s_at NM_020243 740 NP_064628 1485 22 9929 DOWN201751_at NM_014876 741 NP_055691 1486 22 25777 DOWN 212144_at NM_015374742 NP_056189 1487 22 9567 DOWN 205274_at NM_004286; 743 NP_004277; 1488NP_054746 22 9567 DOWN 219357_at NM_004286; 744 NP_004277; 1489NP_054746 22 5155 DOWN 216061_x_at NM_002608; 745; 746 NP_002599; 1490;1491 NM_033016 NP_148937 22 10454 DOWN 203901_at NM_006116; 747; 748NP_006107; 1492; 1493 NM_153497 NP_705717 22 4248 DOWN 208058_s_atNM_002409 749 NP_002400 1494 22 54471 DOWN 221516_s_at NM_019008 750NP_061881 1495 22 54471 DOWN 204593_s_at NM_019008 751 NP_061881 1496 22468 DOWN 200779_at NM_001675; 752; 753 NP_001666; 1497; 1498 NM_182810NP_877962 22 4248 DOWN 209764_at NM_002409 754 NP_002400 1499 22 27352DOWN 203014_x_at NM_015705 755 NP_056520 1500

TABLE 5 Markers of the invention which reside in MCRs of amplificationand display increased expression. Pos Gene Minimum Chromosome (Mb)Weight p value Gene Description Gene Symbol GI UGID# 1 26.8 1.03 0.032nuclear distribution gene C homolog NUDC gi: 5729952 Hs.263812 (A.nidulans) 1 116.9 2.41 0.038 transcription termination factor, RNA TTF2gi: 5733121 Hs.201774 polymerase II 1 117.8 0.85 0.029 WD repeat domain3 WDR3 gi: 5803220 Hs.201375 2 11.3 0.06 0.035 hypothetical proteinMGC33602 MGC33602 gi: 7328008 Hs.274415 5 0.2 0.69 <.005 succinatedehydrogenase complex, SDHA gi: 4759079 Hs.440475 subunit A,flavoprotein (Fp) 5 0.3 0.85 <.005 programmed cell death 6 PDCD6 gi:7019484 Hs.24087 5 0.5 0.95 <.005 Sec 6 (S. cerevisiae) homolog SEC6 gi:3005726 Hs.448580 5 0.6 0.58 0.005 hypothetical protein FLJ10565FLJ10565 gi: 8922520 Hs.100824 5 0.9 0.77 <.005 hypothetical proteinFLJ13441 FLJ13441 gi: 12965190 Hs.449178 5 1.5 0.60 0.014 hypotheticalprotein FLJ12443 FLJ12443 gi: 13376233 Hs.179882 5 1.8 0.96 <.005 NADHdehydrogenase (ubiquinone) Fe—S NDUFS6 gi: 4758791 Hs.408257 protein 6,13 kDa (NADH-coenzyme Q reductase) 5 5.5 0.72 0.005 KIAA0947 proteinKIAA0947 gi: 13436178 Hs.5070 6 32 0.67 0.032 tenascin XB TNXB gi:8361667 Hs.411644 6 42.9 1.03 <.005 trinucleotide repeat containing 5TNRC5 gi: 13325207 Hs.414099 6 43 0.25 0.005 protein phosphatase 2,regulatory subunit PPP2R5D gi: 5453953 Hs.118244 B (B56), delta isoform7 0.8 0.47 0.017 G protein-coupled receptor 30 GPR30 gi: 1381668Hs.113207 7 1.2 0.56 <.005 G protein-coupled receptor 30 GPR30 gi:2656120 Hs.113207 7 1.2 0.14 <.005 DKFZP586J0619 protein DKFZP586J0619gi: 10809392 Hs.112184 7 1.5 0.64 <.005 MICAL-like 2 FLJ23471 gi:13376030 Hs.376617 7 1.6 0.40 0.026 v-maf musculosponeuroticfibrosarcoma MAFK gi: 4505074 Hs.131953 oncogene homolog K (avian) 7 2.10.95 <.005 MAD1 mitotic arrest deficient-like 1 (yeast) MAD1L1 gi:4505064 Hs.7345 7 30.7 0.47 0.027 glycyl-tRNA synthetase GARS gi: 577711Hs.293885 7 64.9 1.19 0.008 argininosuccinate lyase ASL gi: 4502256Hs.442047 7 65 0.87 0.02 calcitonin gene-related peptide-receptor RCP9gi: 7656976 Hs.300684 component protein 7 65.5 1.17 <.005 potassiumchannel tetramerisation domain KCTD7 gi: 5596067 Hs.119683 containing 77 65.6 1.04 <.005 RAB guanine nucleotide exchange factor RABGEF1 gi:7657495 Hs.187660 (GEF) 1 7 65.8 1.42 <.005 hypothetical proteinFLJ10099 FLJ10099 gi: 8922228 Hs.287955 7 93.2 0.69 <.005 BET1 homolog(S. cerevisiae) BET1 gi: 12654162 Hs.23103 7 93.7 0.37 0.05O-acetyltransferase CAS1 gi: 12597638 Hs.324725 7 94.6 0.43 0.032paraoxonase 3 PON3 gi: 1333633 Hs.440967 7 94.6 1.07 <.005 paraoxonase 2PON2 gi: 2228776 Hs.165598 7 94.8 0.50 0.015 pyruvate dehydorgenasekinase, isoenzyme 4 PDK4 gi: 4505692 Hs.8364 7 95.4 0.57 0.017 solutecarrier family 25, member 13 (citrin) SLC25A13 gi: 7657580 Hs.9599 795.9 1.01 <.005 split hand/foot malformation SHFM1 gi: 5453639 Hs.333495(ectrodactyly) type 1 7 96.3 0.42 0.045 ACN9 homolog (S. cerevisiae)ACN9 gi: 9910179 Hs.42785 7 97.1 0.77 <.005 asparagine synthetase ASNSgi: 4502258 Hs.446546 7 97.2 1.23 <.005 Homo sapiens transcribedsequence with gi: 8008445 Hs.512431 weak similarity to protein pir:PC4369 (H. sapiens) PC4369 olfactory receptor. HT2 - human (fragment) 797.3 0.89 <.005 kinase phosphatase inhibitor 2 KPI2 gi: 7662475Hs.122708 7 98.1 0.94 <.005 transformation/transcription domain- TRRAPgi: 4507690 Hs.203952 associated protein 7 98.2 0.66 <.005 E3 ubiquitinligase SMURF1 SMURF1 gi: 4738848 Hs.436249 7 98.2 0.70 <.005transformation/transcription domain- TRRAP gi: 3694662 Hs.203952associated protein 7 98.2 0.58 0.02 E3 ubiquitin ligase SMURF1 SMURF1gi: 6446605 Hs.436249 7 98.2 0.63 0.005 Homo sapiens cDNA: FLJ21284 fis,gi: 10437358 Hs.288218 clone COL01911 7 98.5 0.49 0.013 Homo sapiensclone 24438 mRNA sequence gi: 3283921 Hs.124126 7 98.5 0.99 <.005 actinrelated protein ⅔ complex, subunit ARPC1A gi: 5454077 Hs.291981 1A, 41kDa 7 98.5 1.01 <.005 actin related protein ⅔ complex, subunit ARPC1Bgi: 5031600 Hs.433506 1B, 41 kDa 7 98.7 0.76 <.005 zinc finger protein95 homolog (mouse) ZFP95 gi: 11036641 Hs.110839 7 98.8 0.60 0.005cytochrome P450, family 3, subfamily A, CYP3A5 gi: 945005 Hs.150276polypeptide 5 7 98.8 0.67 <.005 cytochrome P450, family 3, subfamily A,CYP3A5 gi: 4503230 Hs.150276 polypeptide 5 7 99.3 0.59 0.014adaptor-related protein complex 4, mu 1 AP4M1 gi: 5442365 Hs.194703subunit 7 99.3 0.98 <.005 TAF6 RNA polymerase II, TATA box bind- TAF6gi: 5032146 Hs.289950 ing protein (TBP)-associated factor, 80 kDa 7 99.50.38 0.035 postmelotic segregation increased 2-like 6 PMS2L6 gi: 4175684Hs.367667 7 99.8 0.94 0.05 guanine nucleotide binding protein (G GNB2gi: 4885282 Hs.185172 protein), beta polypeptide 2 7 100 0.67 0.005solute carrier family 12 (potassium/chloride SLC12A9 gi: 9910385Hs.437628 transporters), member 9 7 100.4 0.94 <.005 procollagen-lysine,2-oxoglutarate 5- PLOD3 gi: 4505890 Hs.153357 dioxygenase 3 7 100.4 0.500.018 zine finger, HIT domain containing 1 ZNHIT1 gi: 5453616 Hs.2110797 100.4 0.36 0.044 tetratricopeptide repeat domain 11 TTC11 gi: 7705631Hs.423968 7 100.8 0.36 0.047 myosin light chain 2, precursor MYLC2PL gi:12803868 Hs.247831 lymphocyte-specific 7 101.6 0.35 0.007 chromosome 7open reading frame 19 C7orf19 gi: 12357031 Hs.289053 7 101.6 0.49 0.02HSPC047 protein HSPC047 gi: 7661749 Hs.512142 7 101.6 0.36 0.05polymerase (RNA) II (DNA directed) POLR2J gi: 5100572 Hs.489461polypeptide J, 13.3 kDa 7 101.7 0.38 0.038 DNA directed RNA polymeraseII POLR2J2 gi: 10036750 Hs.406505 polypeptide J-related gene 7 101.70.48 <.005 DNA directed RNA polymerase II POLR2J2 gi: 5901957 Hs.406505polypeptide J-related gene 7 105 0.84 <.005 hypothetical proteinMGC33190 MGC33190 gi: 3231718 Hs.211068 7 105.3 0.36 0.03synaptophysin-like protein SYPL gi: 5235354 Hs.80919 7 106.8 0.76 0.048solute carrier family 26, member 4 SLC26A4 gi: 4505696 Hs.512611 7 106.90.48 0.041 Cas-Br-M (murine) ecotropic retroviral CBLL1 gi: 13376203Hs.458382 tranforming sequence-like 1 7 107.1 0.69 0.01 dihydrolipoamidedehydrogenase (E3 DLD gi: 181574 Hs.74635 component of pyruvatedehydrogenase complex, 2-oxo-glutarate complex, branched chain keto aciddehydrogenase complex) 7 111.1 0.50 0.017 dedicator of cytokinesis 4DOCK4 gi: 7662263 Hs.118140 8 37.7 0.60 0.037 G protein-coupled receptor124 GPR124 gi: 11594613 Hs.17270 8 37.7 0.72 0.029 G protein-coupledreceptor 124 GPR124 gi: 4739882 Hs.17270 8 37.7 1.12 <.005 BRF2, subunitof RNA polymerase III BRF2 gi: 11096174 Hs.274136 transcriptioninitiation factor, BRF1-like 8 37.9 0.56 0.008 ash2 (absent, small, orhomeotic)-like ASH2L gi: 4417209 Hs.6856 (Drosophila) 8 38 0.66 0.005LSM1 homolog, U6 small nuclear RNA LSM1 gi: 7657312 Hs.425311 associated(S. cerevisiae) 8 38 0.39 0.046 BCL2-associated athanogene 4 BAG4 gi:6631074 Hs.194726 8 38.1 0.83 <.005 KIAA0725 protein KIAA0725 gi:3882170 Hs.434966 8 38.2 0.46 0.039 Wolf-Hirschhorn syndrome candidate1- WHSC1L1 gi: 13699812 Hs.415895 like 1 8 120.7 0.43 0.026 TAP2 RNApolmerase II, TATA box bind- TAF2 gi: 7022983 Hs.122752 ing protein(TBP)-associated factor, 150 kDa 8 121.3 0.38 0.042 mitochondrialribosomal protein L13 MRPL13 gi: 7662495 Hs.333823 8 122.5 0.43 0.03hysturonan synthase 2 HAS2 gi: 4885390 Hs.159226 8 123.9 0.48 0.014hypothetical protein MGC3067 MGC3067 gi: 8924181 Hs.241576 8 123.9 0.370.05 hypothetical protein MGC3067 MGC3067 gi: 13236515 Hs.241576 8 124.10.44 0.021 unknown MGC21654 product MGC21654 gi: 3231900 Hs.95631 8124.3 0.55 0.008 hypothetical protein FLJ10204 FLJ10204 gi: 8922280Hs.18029 8 124.6 0.52 0.012 annexin A13 ANXA13 gi: 4757753 Hs.181107 8125.4 1.03 <.005 ring finger 139 RNF139 gi: 3395786 Hs.228285 8 125.90.66 <.005 KIAA0196 gene product KIAA0196 gi: 7661987 Hs.437991 8 128.70.62 0.012 v-myc myelocytomatosis viral oncogene MYC gi: 12962934Hs.202453 homolog (avian) 8 128.9 0.62 0.017 Homo sapiens cDNA FLJ26234fis, clone gi: 190753 Hs.459222 ADG09627 8 130.8 0.59 0.013 hypotheticalprotein BM-009 BM-009 gi: 7705303 Hs.369973 8 133.7 0.43 0.022 CGI-72protein CGI-72 gi: 7705782 Hs.44159 8 134.2 0.47 0.023 N-myc downstreamregulated gene 1 NDRG1 gi: 5174656 Hs.318567 8 141.8 0.37 0.048 PTK2protein tyrosine kinase 2 PTK2 gi: 5406748 Hs.434281 8 143.8 0.41 0.034mescochymal stem cell protein DSCD75 LOC51337 gi: 7706199 Hs.25237 8144.6 0.59 0.009 KIAA0150 protein KIAA0150 gi: 1469881 Hs.370491 8 144.70.37 0.037 hypothetical protein FLJ12150 FLJ12150 gi: 13376057 Hs.1189838 144.7 0.37 0.032 eukaryotic translation elongation factor 1 EEF1D gi:4622550 Hs.334798 delta (guanine nucleotide exchange protein) 8 144.80.73 <.005 tissue specific transplantation antigen P35B TSTA3 gi:6598326 Hs.404119 8 144.8 0.80 <.005 tissue specific transplantationantigen P35B TSTA3 gi: 1381178 Hs.404119 8 144.8 0.61 0.01 KIAA0628 geneproduct KIAA0628 gi: 7662213 Hs.43133 8 144.9 0.46 0.022 scribble SCRIBgi: 4331493 Hs.436329 8 145.2 0.61 <.005 5-oxoprolinase(ATP-hydrolysing) OPLAH gi: 5838792 Hs.305882 8 145.2 0.49 0.014 exosomecomplex exonuclease RRP41 RRP41 gi: 4534672 Hs.343589 8 145.2 0.55 0.012exosome complex exonuclease RRP41 RRP41 gi: 9506688 Hs.343589 8 145.20.46 0.021 exosome complex exonuclease RRP41 RRP41 gi: 54085 17Hs.343589 8 145.2 0.37 0.031 GPAA1P anchor attachment protein 1 GPAA1gi: 6031166 Hs.4742 homolog (yeast) 8 145.2 0.51 0.009 GPAA1P anchorattachment protein 1 GPAA1 gi: 13623546 Hs.4742; homolog (yeast); GPAA1Panchor Hs.4742 attachment protein 1 homolog (yeast) 8 145.2 0.45 0.014GPAA1P anchor attachment protein 1 GPAA1 gi: 7018511 Hs.4742 homolog(yeast) 8 145.2 0.43 0.021 cytochrome c-1 CYC1 gi: 4503184 Hs.289271 8145.2 0.39 0.03 hypothetical protein DKFZp434N1923; DKFZP434N1923 gi:13569949 Hs.295866; hypothetical protein DKFZp434N1923 Hs.295866 8 145.30.31 0.014 brain protein 16 LOC51236 gi: 13124772 Hs.300224 8 145.5 0.330.048 diacylglycerol O-acyltransferase homolog DGAT1 gi: 7382489Hs.512810 1 (mouse) 8 145.5 0.67 <.005 putative G-protein coupledreceptor GPCR41 FLJ11856 gi: 13375681 Hs.6459 8 145.6 0.43 0.039cleavage and polyadenylation specific CPSF1 gi: 10037183 Hs.83727 factor1, 160 kDa 8 145.6 0.51 0.01 solute carrier family 39 (zinctransporter), SLC39A4 gi: 8923304 Hs.411274 member 4 8 145.6 0.43 0.031vacuolar protein sorting 28 (yeast) VPS28 gi: 7705884 Hs.418175 8 145.60.44 0.036 Homo sapiens mRNA, chromosome 1 gi: 7150895 Hs.459379specific transcript KIAA0496. 9 21.9 0.02 <.005 methylthioadenosinephosphorylase MTAP gi: 6006025 Hs.446152 9 35.6 0.48 <.005 tropomyosin 2(beta) TPM2 gi: 1219494 Hs.300772 9 35.7 0.52 0.013 talin 1 TLN1 gi:5454129 Hs.375001 9 35.7 0.89 <.005 cAMP responsive element bindingprotein 3 CREB3 gi: 2599559 Hs.287921 9 35.7 0.84 <.005 KIAA0258KIAA0258 gi: 7662029 Hs.47313 9 35.7 0.11 0.032 natriuretic peptidereceptor B/guanylate NPR2 gi: 2337354 Hs.78518 cyclase B(atrionatriuretic peptide receptor B) 9 35.8 0.67 0.006 nasopharyngealcarcinoma related protein NGX6 gi: 7706546 Hs.440953 9 36.1 1.33 <.005clathrin, light polypeptide (Lca) CLTA gi: 6005992 Hs.207052 9 36.2 0.200.005 clathrin, light polypeptide (Lca) CLTA gi: 704460 Hs.207052 9 36.30.49 0.021 ring finger protein 38 RNF38 gi: 12232470 Hs.333503 12 22.10.62 0.006 cytidine monophosphate N- CMAS gi: 8923899 Hs.311346acetylneuraminic acid synthetase 12 50 0.00 0.005 elastase 1, pancreaticELA1 gi: 4503546 Hs.348395 12 54.8 0.77 0.033 myosin, light polypeptide6, alkali, MYL6 gi: 2078957 Hs. 77385 smooth muscle and non-muscle 1322.8 0.84 0.011 ADP-ribosyltransferase (NAD+; poly ADPRTL1 gi: 11496990Hs. 437959 (ADP-ribose) polymerase)-like 1 13 23.6 0.67 0.035myorubularin related protein 6 MTMR6 gi: 1669390 Hs. 79877 13 24.5 2.810.042 ring finger protein (C3H2C3 type) 6 RNF6 gi: 12656362 Hs.136885 13112.9 0.32 0.011 UPF3 regulator of nonsense transcripts UPF3A gi:12620405 Hs.399740 homolog A (yeast) 14 29.1 0.62 0.006 chromosome 14open reading frame 163 C14orf163 gi: 4240322 Hs.27023 14 29.5 0.41 0.038adaptor-related protein complex 4, sigma AP4S1 gi: 12654832 Hs.496614 1subunit 14 30.1 0.80 <.005 chromosome 14 open reading frame 127C14orf127 gi: 13376746 Hs.288981 14 30.6 0.46 0.032 Rho GTPaseactivating protein 5 ARHGAP5 gi: 5905160 Hs.409546 14 31.8 0.56 0.043neuronal PAS domain protein 3 NPAS3 gi: 11545846 Hs.243209 14 32.9 1.120.007 chromosome 14 open reading frame 11 C14orf11 gi: 8922092 Hs.43326914 33 0.98 <.005 sorting nexin 6 SNX6 gi: 13027619 Hs.283443 14 52.41.17 0.013 bone morphogenetic protein 4; bone BMP4 gi: 576934 Hs.68879;morphogenetic protein 4 Hs.68879 14 53.2 0.58 0.021 sterile alpha motifdomain containing 4 SAMD4 gi: 5689442 Hs.98259 14 53.4 0.48 0.025 WDrepeat and HMG-box DNA binding WDHD1 gi: 7704203 Hs.385998 protein 1 1453.5 1.36 <.005 chromosome 14 open reading frame 32 C14orf32 gi:10438139 Hs.406401 14 53.6 0.51 0.013 discs, large homolog 7(Drosophila) DLG7 gi: 7661851 Hs.77695 14 53.8 0.42 0.03 F-box onlyprotein 34 FBXO34 gi: 8923650 Hs.15467 14 54 0.29 0.025 kinectin 1(kinesin receptor) KTN1 gi: 11681348 Hs. 368212 14 55.6 0.62 0.006SEC10-like 1 (S. cerevisiae) SEC10L1 gi: 5730036 Hs. 365863 14 55.7 0.68<.005 chromosome 14 open reading frame 108 C14orf108 gi: 8922687Hs.106210 14 56.6 0.65 0.008 actin-related protein 10 homolog ACTR10 gi:10433604 Hs.248569 (S. cerevisiae) 14 58.5 0.65 0.035 chromosome 14 openreading frame 135 C14orf135 gi: 11968054 Hs.413671 14 59.8 0.62 0.041protein kinase C, eta PRKCH gi: 5453971 Hs.315366 14 60.1 0.76 0.006hypoxia-inducible factor 1, alpha subunit HIF1A gi: 4504384 Hs.412416(basic helix-loop-helix transcription factor) 14 60.2 0.43 0.03 smallnuclear RNA activating complex, SNAPC1 gi: 4507100 Hs. 179312polypeptide 1, 43 kDa 14 62.9 0.49 0.021 zinc finger and BTB domaincontaining 1 ZBTB1 gi: 7662437 Hs. 511938 14 63.2 0.03 0.011 KIAA0599KIAA0599 gi: 13279160 Hs. 198037 14 39.1 0.66 <.005 SWI/SNF related,matrix associated, actin SMARCE1 gi: 13045953 Hs.437546 dependentregulator of chromatin, subfamily e, member 1 17 39.3 1.06 <.005 keratin10 (epidermolytic hyperkeratosis; KRT10 gi: 4557696 Hs.99936 keratosispalmaris et plantaris) 17 40.4 1.74 <.005 ATP citrate lyase ACLY gi:5768107 Hs.387567 17 40.5 0.97 0.007 I-kappa-B-interacting Ras-likeprotein 2 KBRAS2 gi: 8922150 Hs.502910 17 40.6 1.17 0.011 RAB5C, memberRAS occogene family RAB5C gi: 7672664 Hs.479 17 40.7 0.83 0.033 signaltransducer and activator of STAT5B gi: 9970172 Hs.434992 transcription5B 17 41 1.01 <.005 ATPase, H+ transporting, lysosomal V0 ATP6V0A1 gi:4885084 Hs.267871 subunit a isoform 1 17 41 0.69 0.007 transcriptionfactor-like 4 TCFL4 gi: 11761691 Hs.383019 17 41.1 0.89 0.009 GT198,complete ORF HUMGT198A gi: 1164152 Hs.279032 17 41.1 1.06 0.009hypothetical protein LOC162427 LOC162427 gi: 12779367 Hs.432850 17 41.21.30 0.005 enhancer of zeste homolog 1 (Drosophila) EZH1 gi: 2224716Hs.194669 17 41.2 1.08 <.005 enhancer of zeste homolog 1 (Drosophila)EZH1 gi: 2224716 Hs.194669 17 41.3 1.62 <.005 HSPC009 protein HSPC009gi: 7661731 Hs.16059 17 41.3 1.15 <.005 beclin 1 (coiled-coil,myosin-like BCL2 BECN1 gi: 4502394 Hs.12272 interacting protein) 17 41.31.56 <.005 proteasome (prosome, macropain) PSME3 gi: 5031996 Hs.152978activator subunit 3 (PA28 gamma; Ki) 17 41.3 1.09 <.005 amine oxidase,copper containing 2 AOC2 gi: 6806881 Hs.143102 (retina-specific) 17 41.41.74 <.005 hypothetical protein MGC2744 MFC2744 gi: 3432163 Hs.317403 1741.5 1.86 <.005 ribosomal protein L27 RPL27 gi: 4506622 Hs.405528 1741.8 0.44 0.05 Homo sapiens cDNA FLJ35853 fis, clone gi: 5935951Hs.277721 TESTI2007078, highly similar to MEMBRANE COMPONENT, CHROMOSOME17, SURFACE MARKER 2. 17 42.3 0.45 <.005 dual specificity phosphatase 3(vaccinia DUSP3 gi: 12803692 Hs.181046 virus phosphatase VH1-related) 1742.3 1.16 <.005 membrane protein, palmitoylated 3 MPP3 gi: 4505238Hs.396566 (MAGUK p55 subfamily member 3) 17 42.4 0.63 <.005 membraneprotein, palmitoylated 2 MPP2 gi: 6991687 Hs.436326 (MAGUK p55 subfamilymember 2) 17 42.6 1.57 <.005 glucose-6-phosphatase catalytic subunit 3G6PC3 gi: 12951784 Hs.294005 17 42.6 1.39 <.005 glucose-6-phosphatasecatalytic subunit 3 G6PC3 gi: 4834429 Hs.294005 17 42.7 1.55 <.005hypothetical protein MGC3123 MGC3123 gi: 13129117 Hs.181391 17 42.9 0.92<.005 granulin GRN gi: 4504150 Hs.180577 17 42.9 0.45 0.006 KIAA0553protein KIAA0553 gi: 11008117 Hs.396047 17 43.6 0.55 0.018N-myristoyltransferase 1 NMT1 gi: 2760893 Hs.346743 17 43.7 0.34 0.031HMBA-inducible HIS1 gi: 7457641 Hs.15299 17 44 1.09 <.005 hypotheticalprotein FLJ10120 FLJ10120 gi: 8922238 Hs.378860 17 44.8 0.51 0.012 ARFprotein LOC51326 gi: 7770214 Hs.500496 17 44.8 1.09 <.005 KIAA0563 geneproduct KIAA0563 gi: 3647821 Hs.38861 17 45.2 0.92 <.005N-ethylmaleimide-sensitive factor NSF gi: 11079227 Hs.431279 17 45.30.42 0.041 wingless-type MMTV integration site WNT3 gi: 13540476Hs.224667; family, member 3; wingless-type MMTV Hs.224667 integrationsite family, member 3 17 45.7 1.06 <.005 Homo sapiens transcribedsequence with gi: 1126567 Hs.514263 weak similarity to protein sp:P30260 (H. sapiens) CC27_HUMAN Protein CD27Hs (Cell division cycleprotein 27 homolog) (H-NUC) 17 46.1 0.55 0.01 aminopeptidase poromycinsensitive NPEPPS gi: 4210725 Hs.293007 17 46.2 0.31 <.005 karyopherin(importin) beta 1 KPNB2 gi: 13097743 Hs.439683 17 46.4 0.87 0.01pyridoxine-5′-phosphate oxidase PNPO gi: 8922497 Hs.327335 17 46.6 1.27<.005 chromobox homolog 1 (HP1 beta CBX1 gi: 5803075 Hs.77254 homologDrosophila) 17 46.6 0.81 0.008 sorting nexin 11 SNX11 gi: 7019538Hs.15827 17 46.6 0.78 0.01 sorting nexin 11 SNX11 gi: 4827951 Hs.1582717 47.4 0.86 <.005 ATP synthase, H+ transporting, mitochondrial ATP5G1gi: 5262506 Hs.80986 P0 complex, subunit c (subunit 9), isoform 1 1747.4 0.60 0.017 hypothetical protein FLJ13855 FLJ13855 gi: 12751494Hs.369120 17 47.4 1.17 <.005 EAP30 subunit of ELL complex EAP30 gi:6005754 Hs.127249 17 47.8 0.49 0.021 KIAA0924 protein KIAA0924 gi:7662383 Hs.190386 17 47.9 1.15 <.005 prohibitin PHB gi: 6031190 Hs.7532317 48.1 1.06 <.005 specide-type POZ protein SPOP gi: 4507182 Hs.12995117 48.2 1.61 <.005 solute carrier family 35, member B1 SLC35B1 gi:5032212 Hs: 154073 17 48.2 0.82 <.005 C/EBP-induced protein;C/EBP-induced LOC81558 gi: 13540589 Hs.9851; protein Hs.9851 17 48.31.18 <.005 MYST histone acetyltransferase 2 MYST2 gi: 5901961 Hs.2190717 48.6 0.45 0.026 integrin, alpha 3 (antigen CD49C, alpha 3 ITGA3 gi:4504746 Hs.265829 subunit of VLA-3 receptor) 17 48.6 0.37 0.047 pyruvatedehydrogenase kinase, isoenzyme 2 PDK2 gi: 5544583 Hs.92261 17 48.9 0.450.03 xylosyltransferase II XYLT2 gi: 11545913 Hs.32117 17 48.9 0.91<.005 hypothetical protein PRO1855 PRO1855 gi: 10437822 Hs.370927 17 490.79 <.005 hypothetical protein FLJ20920 FLJ20920 gi: 13376740 Hs.28895917 49 0.57 0.017 hypothetical protein FLJ11164 FLJ11164 gi: 8922910Hs.8033 17 49 0.60 0.016 epsin 3 EPN3 gi: 8923677 Hs.165904 17 49.1 0.570.018 hypothetical protein FLJ21347 FLJ21347 gi: 12383067 Hs.103147 1749.2 0.93 <.005 hypothetical protein MGC15396; MGC15396 gi: 13543385Hs.351247; hypothetical protein MGC15396 Hs.351247 17 49.2 0.42 0.034cisplatin resistance-associated LUC7A gi: 7706534 Hs.130293overexpressed protein 17 49.3 0.40 0.041 cisplatin resistance-associatedLUC7A gi: 5174618 Hs.130293 overexpressed protein 17 49.5 0.69 0.017sperm associated antigen 9 SPAG9 gi: 4504524 Hs.500367 17 49.7 1.32<.005 non_metastatic cells 1, protein (NM23A) NME1 gi: 4557796 Hs.118638expressed in 17 49.7 1.75 <.005 non_metastatic cells 2, protein (NM23B)NME2 gi: 4505408 Hs.433416 expressed in 17 49.8 1.01 0.005 CGI-48protein CGI-48 gi: 3179644 Hs.441503 17 53.5 0.61 0.005 COXII homolog,cytochrome c oxidase COXII gi: 4186577 Hs.436988 assembly protein(yeast) 17 53.8 0.23 0.01 hepatic leukemia factor HLF gi: 184223Hs.250692 17 54.3 0.64 0.005 phosphatidylcholine transfer protein PCTPgi: 10864026 Hs.285218 17 74 0.63 0.028 KIAA0195 gene product KIAA0195gi: 7661985 Hs.301132 17 74 1.07 0.009 CASK interacting protein 2CASKIN2 gi: 5766922 Hs.274408 17 74 0.76 0.014 CASK interacting protein2 CASKIN2 gi: 5928096 Hs.274408 18 19.3 0.56 0.022 RIO kinase 3 (yeast)RIOK3 gi: 4507298 Hs.209061 18 19.3 0.79 0.006 RIO kinase 3 (yeast)RIOK3 gi: 5855068 Hs.209061 18 19.3 1.02 <.005 colon cancer-associatedprotein Mic1 MIC1 gi: 7019454 Hs.287633 18 19.3 0.95 <.005 Niemann-Pickdisease, type C1 NPC1 gi: 4557802 Hs.404930 18 19.9 0.48 0.024calcium-binding tyrosine-(Y)- CABYR gi: 6912377 Hs.511983phosphorylation regulated (fibrousheathin 2) 18 20.1 0.48 0.022oxysterol binding protein-like 1A; OSBPL1A gi: 13877169 Hs.415753;oxysterol binding protein-like 1A; Hs.415753 19 41.4 1.18 <.005 zincfinger protein 146 ZNF146 gi: 6005965 Hs.301819 19 42.9 0.71 0.012hypothetical protein FLJ30921 FLJ30921 gi: 9722561 Hs.290703 19 43.30.53 0.028 D4, zinc and double PHD fingers family 1 DPF1 gi: 4758797Hs.389057 19 43.5 1.08 <.005 proteasome (prosome, macropain) 26S PSMD8gi: 4506232 Hs.78466 subunit, non-ATPase, 8 19 43.7 0.54 0.027mitogen-activated protein kinase kinase MAP4K1 gi: 9970929 Hs.95424kinase kinase 1 19 43.8 1.41 <.005 eukaryotic translation initiationfactor 3 eIF3k gi: 5114050 Hs.143773 subunit k 19 43.8 1.43 <.005eukaryotic translation initiation factor 3 eIF3k gi: 6038285 Hs.143773subunit k 19 44 0.43 0.013 heterogeneous nuclear ribonucleoprotein LHNRPL gi: 5935937 Hs.446623 19 44 1.01 <.005 sirtutin (silent matingtype information STRT2 gi: 13775599 Hs.375214 regulation 2 homolog) 2(S. cerevisiae) 19 44 0.70 <.005 nuclear factor of kappa lightpolypeptide NFKBIB gi: 4505384 Hs.9731 gene enhancer in B-cellsinhibitor, beta 19 44.1 0.86 <.005 seryl-tRNA synthetase 2 SARS2 gi:8923420 Hs.14220 19 44.1 0.12 <.005 mitochondrial ribosomal protein S12MRPS12 gi: 2252149 Hs.411125 19 44.1 0.56 0.019 F-box only protein 26FBXO26 gi: 13376364 Hs.425352 19 44.3 0.40 <.005 p21(CDKNIA)-activatedkinase 4 PAK4 gi: 7382497 Hs.20447 19 44.3 0.83 <.005p21(CDKNIA)-activated kinase 4 PAK4 gi: 4101586 Hs.20447 19 44.5 1.16<.005 hypothetical protein F23149_1 PD2 gi: 9506582 Hs.152894 19 50.53.52 0.045 RelA-associated inhibitor RAI gi: 5730000 Hs.324051 19 50.61.27 <.005 CD3-epsilon-associated protein; antisense ASE-1 gi: 6912245Hs.446684 to ERCC-1 19 50.7 3.60 0.045 optic atrophy 3 (autosomalrecessive, with OPA3 gi: 13376716 Hs.123473 chorea and spasticparsplegia) 19 50.8 3.04 0.045 echinoderm microtubule associated EML2gi: 4568182 Hs.24178 protein like 2 19 50.8 0.98 0.018 gastricinhibitory polypeptide receptor GIPR gi: 4503998 Hs.251412 19 50.8 1.000.044 small nuclear ribonucleoprotein D2 SNRPD2 gi: 7242206 Hs.424327polypeptide 16.5 kDa 19 50.9 0.65 0.012 Homo sapiens cDNA FLJ90345 fis,clone gi: 7151592 Hs.43314 NT2RP2002974, highly similar to HOMEOBOXPROTEIN SIX5. 19 50.9 0.49 <.005 dystrophis myotonica-protein kinaseDMPK gi: 189038 Hs.898 19 51.8 0.71 0.005 protein kinase D2 PRKD2 gi:12659006 Hs.205431 19 51.8 0.65 0.009 protein kinase D2 PRKD2 gi:4884153 Hs.205431 19 51.9 1.42 0.008 striatin, calmodulin bindingprotein 4 STRN4 gi: 7019572 Hs.406918 19 51.9 0.90 0.009 solute carrierfamily 1 (neutral amino acid SLC1A5 gi: 4191561 Hs.183556 transporter),member 5 19 52 1.66 <.005 adaptor-related protein complex 2, sigma AP2S1gi: 11038644 Hs.119591 1 subunit 19 52 1.35 <.005 adaptor-relatedprotein complex 2, sigma AP2S1 gi: 13623468 Hs.119591; 1 subunit;adaptor-related protein Hs.119591 complex 2, sigma 1 subunit 19 52.11.87 0.014 glucocorticoid receptor DNA binding GRLF1 gi: 4758481Hs.102548 factor 1 19 52.5 1.05 0.036 complement component 5 receptor 1(C5a C5R1 gi: 4502508 Hs.2161 ligand) 19 52.6 2.86 <.005N-ethylmaleimide-sensitive factor NAPA gi: 4505328 Hs.75932 attachmentprotein, alpha 19 52.9 3.79 0.045 EH-domain containing 2 EHD2 gi:7657053 Hs.325650 19 52.9 5.29 0.045 EH-domain containing 2 EHD2 gi:4261421 Hs.325650 19 52.9 4.35 0.045 EH-domain containing 2 EHD2 gi:4261421 Hs.325650 19 53.3 2.23 0.045 liase 1, DNA, ATP-dependent LIG1gi: 4557718 Hs.1770 19 53.5 1.97 0.045 KDEL (Lys-Asp-Glu-Leu)endoplasmic KDELR1 gi: 5803047 Hs.78040 reticulum protein retentionreceptor 1 19 53.6 3.06 0.045 glutamate-rich WD repeat containing 1GRWD1 gi: 13274610 Hs.400625 19 53.8 1.25 0.041 ribosomal protein L18RPL18 gi: 4834123 Hs.409634 19 53.8 1.82 0.013 D site of albuminpromoter (albumin D- DBP gi: 460704 Hs.414480 box) binding protein 1953.9 0.88 0.033 fucosyltransferase 1 (galactoside 2-alpha- FUT1 gi:4503804 Hs.69747 L-fucosyltransferase) 19 53.9 0.93 0.013fucosyltransferase 1 (galactoside 2-alpha- FUT1 gi: 6739499 Hs.69747L-fucosyltransferase) 19 54 0.58 0.036 pleckstrin homology domaincontaining, PLEKHA4 gi: 10190743 Hs.9469 family A (phosphoinositidebinding specific) member 4 19 54.1 0.43 0.044 nucleobindin 1 NUCB1 gi:5453817 Hs.172609 19 54.1 0.63 0.032 BCL2-associated X protein BAX gi:4751837 Hs.159428 19 54.1 1.36 <.005 glycogen synthase 1 (muscle) GYS1gi: 4504232 Hs.386225 19 54.1 1.13 <.005 RuvB-like 2 (E. coli) RUVBL2gi: 5730022 Hs.6455 19 54.2 0.94 0.011 luteinizing hormone betapolypeptide LHB gi: 4504988 Hs.154704 19 54.2 2.38 <.005 small nuclearribonucleoprotein 70 kDa SNRP70 gi: 4507118 Hs.174051 polypeptide (RNPantigen) 19 54.5 0.95 0.011 soggy-1 gene DKKL1-pending gi: 7657553Hs.124021 19 54.6 0.51 0.034 ribosomal protein L13a RPL13A gi: 12653484Hs.449070 19 54.7 0.92 0.029 reticulocalbin 3, EF-hand calcium bindingRCN3 gi: 10257434 Hs.439184 domain 19 54.7 0.99 0.012 nitric oxidesynthase interacting protein NOSIP gi: 7705715 Hs.7236 19 54.8 0.650.017 interferon regulatory factor 3 IRF3 gi: 4504724 Hs.75254 19 54.82.10 0.045 HMT1 hnRNP methyltransferase-like 2 HRMT1L2 gi: 4504496Hs.20521 (S. cerevisiae) 19 55 1.21 0.014 prostate tumor overexpressedgene 1 PTOV1 gi: 4884338 Hs.227429 19 55.1 2.89 0.045 nucleoporin 62 kDaNUP62 gi: 7705354 Hs.437023 19 55.5 0.48 0.024 nuclear receptorsubfamily 1, group H, NR1H2 gi: 11321629 Hs.432976 member 2 19 56.2 0.110.007 kallikrein 13 KLK13 gi: 4884461 Hs.165296 19 58.7 1.51 <.005 zincfinger protein 331 ZNF331 gi: 10092612 Hs.147644 19 59.3 1.45 <.005CCR4-NOT transcription complex, subunit 3 CNOT3 gi: 7657386 Hs.343571 1959.3 0.87 0.013 leukocyte receptor cluster (LRC) member 4 LENG4 gi:13236521 Hs.78768 16 59.3 0.65 0.013 leukocyte receptor cluster (LRC)member 5 LENG5 gi: 13129061 Hs.15580 19 59.4 0.73 0.019 ribosomalprotein S9 RPS9 gi: 4506744 Hs.139876 19 60.3 0.50 0.018 synaptotagmin VSVT5 gi: 4763527 Hs.23179 19 60.8 1.68 0.01 LDL induced EC proteinLOC51157 gi: 7705880 Hs.94392 19 60.8 0.88 0.019 U2 (RNU2) small nuclearRNA auxiliary U2AF2 gi: 5396722 Hs.297629 factor 2 19 60.8 1.08 0.027epsin 1 EPN1 gi: 7019368 Hs.279953 19 61.2 1.42 <.005 hypotheticalprotein LOC126208 LOC126108 gi: 1496048 Hs.397153 19 61.3 0.55 0.039zinc finger protein 444 ZNF444 gi: 8922893 Hs.24545 19 61.3 0.62 0.027zinc finger protein 444 ZNF444 gi: 3916863 Hs.24545 19 62.4 0.69 0.022zinc finger protein 264 ZNF264 gi: 4585642 Hs.426358 19 62.4 0.79 <.005zinc finger protein 272 ZNF272 gi: 498733 Hs.99971 19 62.5 0.44 0.043zinc finger protein 304 ZNF304 gi: 10190695 Hs.287374 19 62.6 0.66 0.008hypothetical protein FLJ23233 FLJ23233 gi: 13375967 Hs.98593 19 62.60.60 0.023 hypothetical protein FLJ23233 FLJ23233 gi: 2112716 Hs.9859319 62.8 0.60 0.022 zinc finger protein 134 (clone pHZ-15) ZNF134 gi:4507982 Hs.449971 19 62.8 1.08 <.005 zinc finger protein 211 ZNF211 gi:5454175 Hs.449970 20 30.9 0.62 0.008 inhibitor of DNA binding 1,dominant ID1 gi: 464181 Hs.410900 negative helix-loop-helix protein 2058.2 0.70 <.005 ATP synthase, H+ transporting, ATP5E gi: 5901895Hs.177530 mitochondrial F1 complex, epsilon subunit 20 58.2 0.65 <.005chromosome 20 open reading frame 45 C20orf45 gi: 7705609 Hs.3945 20 59.20.68 <.005 protein phosphatase 1, regulatory subunit 3D PPP1R3D gi:6806895 Hs.504920 20 61.2 0.00 0.025 TAF4 RNA polymerase II, TATA boxbind- TAF4 gi: 5112317 Hs.24644 ing protein (TBP)-associated factor. 135kDa 20 61.4 0.45 0.015 synovial sarcoma translocation gene on SS18L1 gi:3327199 Hs.154429 chromosome 18-like 1 20 61.4 0.38 0.048 proteasome(prosome, macropain) PSMA7 gi: 9408092 Hs.233952 subunit, alpha type, 720 61.5 0.59 <.005 oxysterol binding protein-like 2 OSBPL2 gi: 12653062Hs.15519 20 61.5 0.37 0.049 laminin, alpha 5 LAMA5 gi: 13097167 Hs.1166920 61.6 0.06 <.005 ribosomal protein S21 RPS21 gi: 4506698 Hs.372960 2062.2 0.49 0.018 transcription factor-llke 5 (basic helix- TCFL5 gi:5730082 Hs.30696 loop-helix) 20 62.2 0.39 0.03 chromosome 20 openreading frame 11 C20orf11 gi: 8923556 Hs.103808 20 62.5 0.82 <.005chromosome 20 open reading frame 21 C20orf21 gi: 9663381 Hs.11747 20 630.46 0.018 ADP-ribosylation factor related protein 1 ARFRP1 gi: 8246778Hs.389277 20 63 0.41 0.032 KIAA1847 KIAA1847 gi: 8246778 Hs.11900 20 630.37 0.03 ADP-ribosylation factor related protein 1 ARFRP1 gi: 4507448Hs.389277 20 63 0.48 0.016 KIAA1847 KIAA1847 gi: 2279318 Hs.11900 2063.2 0.77 <.005 tumor protein D52-like 2 TPD52L2 gi: 4507642 Hs.15471820 63.3 0.73 <.005 uridine kinase-like 1 URKL1 gi: 8923486 Hs.504998 2063.3 0.75 <.005 chromosome 20 open reading frame 14 C20orf14 gi:13401215 Hs.31334 22 22.6 0.59 0.031 D-dopachrome tautomerase DDT gi:5453630 Hs.433902 22 22.7 0.42 0.018 glutathione S-transferase theta 1GSTT1 gi: 4504184 Hs.268573 22 29.6 0.46 0.015 hypothetical proteinFLJ20618 FLJ20618 gi: 7021031 Hs.52184 22 29.6 0.65 <.005 hypotheticalprotein FLJ20618 FLJ20618 gi: 4899032 Hs.52184 Ref Seq SEQ ID NO.: RefSeq SEQ ID NO.: Chromosome Locus Link Regulation Probes mRna ID Nuc.Prot ID A.A. 1 10726 UP 201173_x_at NM_006600 445 NP_006591 1196 1 8458UP 204407_at NM_003594 1 NP_003585 756 1 10885 UP 218882_s_at NM_0067842 NP_006775 757 2 130814 UP 216458_at NM_152391 458 NP_689604 1209 56389 UP 201093_x_at NM_004168 3 NP_004159 758 5 10016 UP 203415_atNM_013232 4 NP_037364 759 5 11336 UP 212630_at NM_007277 5 NP_009208 7605 55722 UP 219531_at NM_018140 6 NP_060610 761 5 65980 UP 220155_s_atNM_023924 7 NP_076413 762 5 79888 UP 201818_at NM_024830 8 NP_079106 7635 4726 UP 203606_at NM_004553 9 NP_004544 764 5 23379 UP 209654_atXM_029101 10 XP_029101 765 6 7148 UP 213451_x_at NM_019105; 11; 12NP_061978; 766; 767 NM_032470 NP_115859 6 10695 UP 217931_at NM_006586;13; 14 NP_006577; 768; 769 NM_183010 NP_898828 6 5528 UP 202513_s_atNM_006245; 15; 16; 17 NP_006236; 770; 771; 772 NM_180976; NP_851307;NM_180977 NP_851308 7 2852 UP 211829_s_at NM_001505 18 NP_001496 773 72852 UP 210640_s_at NM_001505 19 NP_001496 774 7 26173 UP 212212_s_atXM_291222 20 XP_291222 775 7 79778 UP 219332_at NM_024723; 21; 22NP_078999; 776; 777 NM_182924 NP_891554 7 7975 UP 206750_at NM_002360 23NP_002351 778 7 8379 UP 204857_at NM_003550 24 NP_003541 779 7 2617 UP208693_s_at NM_002047 25 NP_002038 780 7 435 UP 204608_at NM_000048 26NP_000039 781 7 27297 UP 203899_s_at NM_014478 27 NP_055293 782 7 154881UP 213474_at NM_153033 28 NP_694578 783 7 27342 UP 218310_at NM_01450429 NP_055319 784 7 55069 UP 218008_at NM_017994 30 NP_060464 785 7 10282UP 202710_at NM_005868 31 NP_005859 786 7 64921 UP 219342_at NM_02290032 NP_075051 787 7 5446 UP 213695_at NM_000940 33 NP_000931 788 7 5445UP 210830_s_at NM_000305 34 NP_000296 789 7 5166 UP 205960_at NM_00261235 NP_002603 790 7 10165 UP 203775_at NM_014251 36 NP_055066 791 7 7979UP 202276_at NM_006304 37 NP_006295 792 7 57001 UP 218981_at NM_02018638 NP_064571 793 7 440 UP 205047_s_at NM_001673; 39; 40; 41 NP_001664;794; 795; 796 NM_133436; NP_597680; NM_183356 NP_899199 7 UP 217499_x_at1516 7 22853 UP 206223_at NM_014916 42 NP_055731 797 7 8295 UP20642_s_at NM_003496 43 NP_003487 798 7 57154 UP 212668_at NM_020429;44; 45 NP_065162; 799; 800 NM_181349 NP_851994 7 8295 UP 214908_a_atNM_003496 46 NP_003487 801 7 57154 UP 215458_s_at NM_020429; 47; 48NP_065162; 802; 803 NM_181349 NP_851994 7 UP 215589_at 1517 — 7 UP215457_at 1518 — 7 10552 UP 200950_at NM_006409 49 NP_006400 804 7 10095UP 201954_at NM_005720 50 NP_005711 805 7 23600 UP 203731_s_atNM_014569; 51; 52 NP_055384; 806; 807 NM_145102 NP_659570 7 1577 UP214234_s_at NM_000777 53 NP_000768 808 7 1577 UP 205765_at NM_000777 54NP_000768 809 7 9179 UP 209837_at NM_004722 55 NP_004713 810 7 6878 UP203572_s_at NM_005641; 56; 57; 58; 59 NP_005632; 811; 812; 813; 814NM_139122; NP_620834; NM_139123; NP_620835; NM_139315 NP_647476 7 5384UP 215667_x_at 1519 — 7 2783 UP 200852_x_at NM_005273 60 NP_005264 815 756996 UP 220371_s_at NM_020246 61 NP_064631 816 7 8985 UP 202185_atNM_001084 62 NP_001075 817 7 10467 UP 201541_s_at NM_006349 63 NP_006340818 7 51024 UP 218034_at NM_016068 64 NP_057152 819 7 93408 UP221659_s_at NM_138403 65 NP_612412 820 7 80228 UP 218811_at NM_032831 66NP_079432; 821 NP_116220 7 29060 UP 220692_at NM_014147 67 NP_054866 8227 5439 UP 212707_s_at NM_006234 68 NP_006225 823 7 246721 UP 214740_atNM_032958; 69; 70; 71 NP_116580; 824; 825; 826 NM_032959; NP_116581;NM_145325 NP_663165 7 246721 UP 208534_s_at NM_032958; 72; 73; 74NP_116580; 827; 828; 829 NM_032959; NP_116581; NM_145325 NP_663165 7222255 UP 214342_at NM_152749 75 NP_689962 830 7 6856 UP 201259_s_atNM_006754; 76; 77 NP_006745; 831; 832 NM_182715 NP_874384 7 5172 UP206529_x_at NM_000441 78 NP_000432 833 7 79872 UP 220018_at NM_024814 79NP_079090 834 7 1738 UP 209095_at NM_000108 80 NP_000099 835 7 9732 UP205003_at NM_014705 81 NP_055520 836 8 25960 UP 221814_at NM_032777 82NP_116166 837 8 25960 UP 65718_at NM_032777 83 NP_116166 838 8 55290 UP218954_s_at NM_018310 84 NP_060780 839 8 9070 UP 209517_s_at NM_00467485 NP_004665 840 8 27257 UP 203534_at NM_014462 86 NP_055277 841 8 9530UP 219624_at NM_004874 87 NP_004865 842 8 23259 UP 212690_at NM_29129188 XP_291291 843 8 54904 UP 218173_s_at NM_017778; 89; 90 NP_060248;844; 845 NM_023034 NP_075447 8 6873 UP 209523_at NM_003184 91 NP_003175846 8 28998 UP 218049_s_at NM_014078 92 NP_054797 847 8 3037 UP206432_at NM_005328 93 NP_005319 848 8 79139 UP 218172_s_at NM_024295 94NP_061100; 849 NP_077271 8 79139 UP 219402_s_at NM_024295 95 NP_061100;850 NP_077271 8 93594 UP 214061_at NM_145647 96 NP_663622 851 8 55093 UP219060_at NM_018024 97 NP_060494 852 8 312 UP 208323_s_at NM_004306 98NP_004297 853 8 11236 UP 209510_at NM_007218 99 NP_009149 854 8 9897 UP201985_at NM_014846 100 NP_055661 855 8 4609 UP 202431_s_at NM_002467101 NP_002458 856 8 375682 UP 216240_at 1520 — 8 51571 UP 217916_s_atNM_016623 102 NP_057707 857 8 51105 UP 219606_at NM_016018; 103; 104;105; 106 NP_057102; 858; 859; 860; 861 NM_024878; NP_079154; NM_032205;NP_115581; NM_198513 NP_940915 8 10397 UP 200632_s_at NM_006096 107NP_006087 862 8 5747 UP 208820_at NM_005607; 108; 109 NP_005598; 863;864 NM_153831 NP_722560 8 51337 UP 218500_at NM_016647 110 NP_057731 8658 23144 UP 213445_at NM_015117 111 NP_055932 866 8 79792 UP 218154_atNM_024736 112 NP_079012 867 8 1936 UP 214394_x_at NM_001960; 113; 114NP_001951; 868; 869 NM_032378 NP_115754 8 7264 UP 201644_at NM_003313115 NP_003304 870 8 7264 UP 36936_at NM_003313 116 NP_003304 871 8 9831UP 206188_at 1521 1522 8 23513 UP 212556_at NM_015356; 117 NP_056171;872; 873 NM_182706 NP_874365 8 26873 UP 222025_s_at XM_291266 118XP_291266 874 8 54512 UP 91682_at NM_019037 119 NP_061910 875 8 54512 UP218695_at NM_019037 120 NP_061910 876 8 54512 UP 58696_at NM_019037 121NP_061910 877 8 8733 UP 201618_x_at NM_003801 122 NP_003792 878 8 8733;8733 UP 211060_x_at NM_003801 123 NP_003792 879 8 8733 UP 215690_x_atNM_003801 124 NP_003792 880 8 1537 UP 201066_at NM_001916 125 NP_001907881 8 81858; UP 220973_s_at NM_030974 126 NP_112236 882 81858 8 51236 UP219071_x_at NM_016458 127 NP_057542 883 8 8694 UP 203669_s_at NM_012079128 NP_036211 884 8 79581 UP 218151_x_at NM_024531 129 NP_078807 885 829894 UP 201638_s_at NM_013291 130 NP_037423 886 8 55630 UP 219215_s_atNM_017767; 131; 132 NP_060237; 887; 878 NM_130849 NP_570901 8 51160 UP218679_s_at NM_016208; 133; 134 NP_057292; 879; 880 NM_183057 NP_8988808 UP 213681_at 1523 — 9 4507 UP 204956_at NM_002451 555 NP_002442 1303 97169 UP 212654_at NM_003289 135 NP_003280 891 9 7094 UP 203254_s_atNM_006289 136 NP_006280 892 9 10488 UP 209432_s_at NM_006368 137NP_006359 893 9 9827 UP 203169_at XM_376830 138 XP_376830 894 9 4882 UP214066_x_at NM_000907; 139; 140 NP_000898; 895 NM_003995 NP_003986 951754 UP 207839_s_at NM_016446 141 NP_057530 896 9 1211 UP 200960_x_atNM_001833; 142; 143 NP_001824; 897; 898 NM_007096 NP_009027 9 1211 UP216293_at NM_001833; 144; 145 NP_001824; 899; 900 NM_007096 NP_009027 9152006 UP 218528_s_at NM_022781; 146; 147; 148; 149; NP_073618; 901;902; 903; 904; NM_194328; 150; 151 NP_919309; 905; 906 NM_194329;NP_919310; NM_194330; NP_919311; NM_194331; NP_919312; NM_194332NP_919313 12 55907 UP 218111_s_at NM_018686 152 NP_061156 907 12 1990 UP206446_s_at NM_001971 586 NP_001962 1331 12 4637 UP 214002_at NM_021019;610; 611; 612; 613 NP_066299; 1355; 1356; 1357; NM_079423; NP_524147;1358 NM_079424; NP_524148; NM_079425 NP_524149 13 143 UP 202239_atNM_006437 153 NP_006428 908 13 9107 UP 214429_at NM_004685 154 NP_004676909 13 6049 UP 210931_at NM_005977; 155; 156; 157; 158 NP_005968; 910;911; 912; 913 NM_183043; NP_898864; NM_183044; NP_898865; NM_183045NP_898866 13 65110 UP 206958_s_at NM_023011; 159; 160 NP_075387; 914;915 NM_080687 NP_542418 14 23256 UP 215548_s_at NM_016106; 161; 162NP_057190; 916; 917 NM_182835 NP_878255 14 11154 UP 210952_at NM_007077163 NP_009008 918 14 80224 UP 220176_at NM_025152 164 NP_079428 919 14394 UP 217936_at NM_001173 165 NP_001164 920 14 64067 UP 220316_atNM_022123; 166; 167 NP_071406; 921; 922 NM_173159 NP_775182 14 55837 UP202623_at NM_018453 168 NP_060923 923 14 58533 UP 217789_at NM_021249;169; 170 NP_067072; 924; 925 NM_152233 NP_689419 14 652; 652 UP211518_s_at NM_001202; 171; 172; 173; 174; NP_001193; 926; 927; 928NM_130850; 175; 176 NP_570911; NM_130851; NP_570912 NM_001202;NM_130850; NM_130851 14 23034 UP 212845_at NM_015589 177 NP_056404 92914 11169 UP 204727_at NM_007086 178 NP_009017 930 14 93487 UP212499_s_at NM_144578 179 NP_653179 931 14 9787 UP 203764_at NM_014750180 NP_055565 932 14 55030 UP 218539_at NM_017943 181 NP_060413 933 143895 UP 200914_x_at NM_182926 182 NP_891556 934 14 10640 UP 218748_s_atNM_006544 183 NP_006535 935 14 55745 UP 218139_s_at NM_018229 184NP_060699 936 14 55860 UP 222230_s_at NM_018477 185 NP_060947 937 1464430 UP 219972_s_at NM_022495 186 NP_071940 938 14 5583 UP 206099_atNM_006255; 187 NP_006246; 939 NP_076969 14 3091 UP 200989_at NM_001530;188; 189 NP_001521; 940; 941 NM_181054 NP_851397 14 6617 UP 205443_atNM_003082 190 NP_003073 942 14 22890 UP 205092_x_at XM_375086 191XP_375086 943 14 26030 UP 217044_s_at 1524 1525 14 6605 UP 211988_atNM_003079 192 NP_003070 944 17 3858 UP 207023_x_at NM_000421 193NP_000412 945 17 47 UP 201127_s_at NM_001096; 194; 195 NP_001087; 946NM_198830 NP_942127 17 28511 UP 218240_at NM_017595 196 NP_060065 947 175878 UP 201156_s_at NM_004583; 197; 198 NP_004574; 948; 949 NM_201434NP_958842 17 6777 UP 212549_at NM_012448 199 NP_036580 950 17 535 UP205095_s_at NM_005177 200 NP_005168 951 17 6945 UP 210752_s_atNM_170607; 201; 202; 203 NP_733752; 952; 953; 954 NM_198204; NP_937847;NM_198205 NP_937848 17 29893 UP 213708_s_at NM_013290; 204; 205NP_037422; 955; 956 NM_016556 NP_057640 17 162427 UP 212697_at NM_178126206 NP_835227 957 17 2145 UP 203249_at NM_001991 207 NP_001982 958 172145 UP 32259_at NM_001991 208 NP_001982 959 17 28958 UP 218026_atNM_014019 209 NP_054738 960 17 8678 UP 208945_s_at NM_003766 210NP_003757 961 17 10197 UP 200988_s_at NM_005789; 211; 212 NP_005780;962; 963 NM_176863 NP_789839 17 314 UP 207064_s_at NM_001158; 213; 214NP_001149; 964; 965 NM_009590 NP_0033720 17 80755 UP 222064_s_atNM_025267 215 NP_079543 966 17 6155 UP 200025_s_at NM_000988 216NP_000979 967 17 UP 201383_s_at 1526 17 1845 UP 201537_s_at NM_004090217 NP_004081 968 17 4356 UP 206186_at NM_001932 218 NP_001923 969 174355 UP 213270_at NM_005374 219 NP_005365 970 17 92579 UP 221759_atNM_138387 220 NP_612396 971 17 92579 UP 44654_at NM_138387 221 NP_612396972 17 79089 UP 218419_s_at NM_024107; 222; 223 NP_077012; 973; 974NM_17741 NP_803190 17 2896 UP 200678_x_at NM_002087 224 NP_002078 975 1723131 UP 212485_at XM_290758 225 XP_290758 976 17 4836 UP 201157_s_atNM_021079 226 NP_066565 977 17 10614 UP 214188_at NM_006460 227NP_006451 978 17 55073 UP 220219_s_at NM_018001 228 NP_060471 979 1751326 UP 210718_s_at NM_016632 229 NP_057716 980 17 9884 UP 221740_x_atNM_014834 230 NP_055649 981 17 4905 UP 202395_at NM_006178 231 NP_006169982 17 7473; 7473 UP 221455_s_at NM_030753 232 NP_110380 983 17 UP217880_at 1527 — 17 9520 UP 201455_s_at NM_006310 235 NP_006301 986 173837 UP 208974_x_at NM_002265 236 NP_002256 987 17 55163 UP 218511_s_atNM_018129 237 NP_060599 988 17 10951 UP 201518_at NM_006807 238NP_006798 989 17 29916 UP 220140_s_at NM_013323; 239; 240 NP_037455;990; 991 NM_152244 NP_689450 17 29916 UP 53912_at NM_013323; 241; 242NP_689450 992; 993 NM_152244 NP_689450 17 516 UP 208972_s_at NM_005175243 NP_005166 994 17 65264 UP 217750_s_at NM_023079 244 NP_075567 995 1711267 UP 218391_at NM_007241 245 NP_009172 996 17 22834 UP 205594_atXM_375471 246 XP_375471 997 17 5245 UP 200659_s_at NM_002634 247NP_002625 998 17 8405 UP 204640_s_at NM_003563 248 NP_003554 999 1710237 UP 202433_at NM_005827 249 NP_005818 1000 17 81558; UP 221249_s_atNM_030802 250 NP_110429 1001 81558 17 11143 UP 200049_at NM_007067 251NP_008998 1002 17 3675 UP 201474_s_at NM_002204; 252; 253 NP_002195;1003; 1004 NM_005501 NP_005492 17 5164 UP 213724_s_at NM_002611 254NP_002602 1005 17 64132 UP 219401_at NM_022167 255 NP_071450 1006 1755379 UP 222231_s_at NM_018509 256 NP_060979 1007 17 80221 UP 218844_atNM_025149 257 NP_079425 1008 17 55316 UP 218307_at NM_018346 258NP_060816 1009 17 55040 UP 220318_at NM_017957 259 NP_060427 1010 1764847 UP 218164_at NM_022827 260 NP_073738 1011 17 91369; UP 211717_atNM_052855 261 NP_443087 1012 91369 17 51747 UP 220044_x_at NM_016424 262NP_057508 1013 17 51747 UP 203804_s_at NM_016424 263 NP_057508 1014 179043 UP 206748_s_at NM 003971, 264; 265 NP_003962; 1015; 1016 NM_172345NP_758853 17 4830 UP 201577_at NM_000269; 266; 267 NP_000260; 1017; 1018NM_198175 NP_937818 17 4831 UP 201268_at NM_002512 268 NP_002503 1019 1751096 UP 222038_s_at NM_016001 269 NP_057085 1020 17 1353 UP 214277_atNM_004375 270 NP_004366 1021 17 3131 UP 204755_x_at NM_002126 271NP_002117 1022 17 58488 UP 218676_s_at NM_021213 272 NP_067036 1023 179772 UP 202650_s_at NM_014738 273 NP_055553 1024 17 57513 UP 221846_s_atNM_020753 274 NP_065804 1025 17 57513 UP 61297_at NM_020753 275NP_065804 1026 18 8780 UP 202131_s_at NM_003831; 276; 277 NP_003822;1027; 1028 NM_145906 NP_665913 18 8780 UP 202129_s_at NM_003831; 278;279 NP_003822; 1029; 1030 NM_145906 NP_665913 18 29919 UP 221190_s_atNM_013326 280 NP_037458 1031 18 4864 UP 202679_at NM_000271 281NP_000262 1032 18 26256 UP 219928_s_at NM_012189; 282; 283; 284; 285;NP_036321; 1033; 1034; 1035; NM_138643; 286; 287 NP_619584; 1036; 1037;1038 NM_138644; NM_619585; NM_153768; NP_722452; NM_153769; NP_722453;NM_153770 NP_722454 18 114876; UP 208158_s_at NM_018030; 288; 289; 290NP_060500; 1039; 1040; 1041 114876 NM_080597; NP_542164; NM_133268NP_579802 19 7705 UP 200050_at NM_007145 291 NP_009076 1042 19 126231 UP217627_at NM_152360 292 NP_689573 1043 19 8193 UP 206531_at NM_004647293 NP_004638 1044 19 5714 UP 200820_at NM_002812 294 NP_002803 1045 1911184 UP 214219_x_at NM_007181 295 NP_009112 1046 19 27335 UP221494_x_at NM_013234 296 NP_037366 1047 19 27335 UP 212716_s_atNM_013234 297 NP_037366 1048 19 3191 UP 221860_at NM_001533 298NP_001524 1049 19 22933 UP 220605_s_at NM_012237; 299; 300 NP_036369;1050; 1051 NM_030593 NP_085096 19 4793 UP 214448_x_at NM_002503 301NP_002494 1052 19 54938 UP 218702_at NM_017827 302 NP_060297 1053 196183 UP 210008_s_at NM_021107; 303; 304; 305 NP_066930; 1054; 1055; 1056NM_033362; NP_203526; NM_033363 NP_203527 19 115290 UP 220233_atNM_024907; 306; 307 NP_079183; 1057; 1058 NM_148169 NP_680474 19 10298UP 203154_s_at NM_005884 308 NP_005875 1059 19 10298 UP 33814_atNM_005884 309 NP_005875 1060 19 54623 UP 202093_s_at NM_019088 310NP_061961 1061 19 10848 UP 218849_s_at NM_006663 311 NP_006654 1062 1910849 UP 205264_at NM_012099 312 NP_036231 1063 19 80207 UP 206357_atNM_025136 313 NP_079412 1064 19 24139 UP 204399_s_at NM_012155 314NP_036287 1065 19 2696 UP 208105_at NM_000164 315 NP_000155 1066 19 6633UP 200826_at NM_004597; 316; 317 NP_004588; 1067; 1068 NM_177542NP_808210 19 UP 217661_x_at 1528 19 1760 UP 217062_at NM_004409 318NP_004400 1069 19 25865 UP 209282_at NM_016457 320 NP_057541 1071 1925865 UP 38269_at NM_016457 321 NP_057541 1072 19 29888 UP 217903_atNM_013403 322 NP_037535 1073 19 6510 UP 208916_at NM_005628 323NP_005619 1074 19 1175 UP 202120_x_at NM_004069; 324; 325 NP_004060;1075; 1976 NM_021575 NP_067586 19 1175; 1175 UP 211047_x_at NM_004069;326; 327 NP_004060; 1077; 1078 NM_021575 NP_067586 19 2909 UP202046_s_at NM_004491; 328; 329 NP_004482; 1079; 1080 NM_024342NP_077318 19 728 UP 220088_at NM_001736 330 NP_001727 1081 19 8775 UP206491_s_at NM_003827 331 NP_003818 1082 19 30846 UP 205341_at NM_014601332 NP_055416 1083 19 30846 UP 221870_at NM_014601 333 NP_055416 1084 1930846 UP 45297_at NM_014601 334 NP_055416 1085 19 3978 UP 202726_atNM_000234 335 NP_000225 1086 19 10945 UP 200922_at NM_006801 336NP_006792 1087 19 83743 UP 221549_at NM_031485 337 NP_113673 1088 196141 UP 214335_at NM_000979 338 NP_000970 1089 19 1628 UP 209783_atNM_001352 339 NP_001343 1090 19 2523 UP 206109_at NM_000148 340NP_000139 1091 19 2523 UP 211411_at NM_000148 341 NP_000139 1092 1957664 UP 219011 _at NM_020904 342 NP_065955 1093 19 4924 UP 200646_s_atNM_006184 343 NP_006175 1094 19 581 UP 208478_s_at NM_004324; 344; 345;346; 347; NP_004315; 1095; 1096; 1097; NM_138761; 348; 349 NP_620116;1098; 1099; 1100 NM_138762; NP_620117; NM_138763; NP_620118; NM_138764;NP_620119; NM_138765 NP_620120 19 2997 UP 201673_s_at NM_002103 350NP_002094 1101 19 10856 UP 201459_at NM_006666 351 NP_006657 1102 193972 UP 214471_x_at NM_000894 352 NP_000885 1103 19 6625 UP 201221_s_atNM_003089 353 NP_003080 1104 19 27120 UP 220284_at NM_014419 354NP_055234 1105 19 23521 UP 200715_x_at NM_012423 355 NP_036555 1106 1957333 UP 219102_at NM_020650 356 NP_065701 1107 19 51070 UP 217950_atNM_015953 357 NP_057037 1108 19 3661 UP 202621_at NM_001571 358NP_001562 1109 19 3276 UP 206445_s_at NM_001536: 359; 360; 361NP_001527; 1110; 1111; 1112 NM_198318; NP_938074; NM_198319 NP_938075 1953635 UP 213690_s_at NM_017432 362 NP_059128 1113 19 23636 UP202153_s_at NM_012346; 363; 364; 365; 366 NP_036478; 1114; 1115; 1116;NM_016553; NP_057637; 1117 NM_153718: NP_714940: NM_153719 NP_714941 197376 UP 218215_s_at NM_007121 367 NP_009052 1118 19 26085 UP 216670_atNM_015596 368 NP_056411 1119 19 55422 UP 219228_at NM_018555 369NP_061025 1120 19 4849 UP 203239_s_at NM_014516 371 NP_055331 1122 1979143 UP 205634_x_at NM_024298 372 NP_077274 1123 16 79042 UP218132_s_at NM_024075 373 NP_076980 1124 19 6203 UP 217747_s_atNM_001013 374 NP_001004 1125 19 6861 UP 206161_s_at NM_003180 375NP_003171 1126 19 51157 UP 220748_s_at NM_016202 376 NP_057286 1127 1911338 UP 214171_s_at NM_007279 377 NP_009210 1128 19 29924 UP221141_x_at NM_013333 378 NP_037465 1129 19 126208 UP 213402_atXM_058999 379 XP_058999 1130 19 55311 UP 218707_at NM_018337 380NP_060807 1131 19 55311 UP 50376_at NM_018337 381 NP_060807 1132 19 9423UP 205917_at XM_375660 382 XP_375660 1133 19 10794 UP 216273_atNM_006635 383 NP_006626 1134 19 57343 UP 207753_at NM_020657 384NP_065708 1135 19 79744 UP 219826_at NM_024691 385 NP_078967 1136 1979744 UP 58367_s_at NM_024691 386 NP_078967 1137 19 7693 UP 206182_atNM_003435 387 NP_003426 1138 19 10520 UP 205437_at NM_006385: 388; 389NP_0063763 1139; 1140 NM_198855 NP_942152 20 3397 UP 208937_s_atNM_002165; 390; 391 NP_002156; 1141, 1142 NM_181353 NP_851998 20 514 UP217801_at NM_006886 392 NP_008817 1143 20 51012 UP 217851_s_at NM_016045393 NP_057129 1144 20 5509 UP 204555_s_at NM_006242 394 NP_006233 114520 6874 UP 213090_s_at NM_003185 395 NP_003176 1146 20 26039 UP213140_s_at NM_015558; 396; 397 NP_056373; 1147; 1148 NM_198935NP_945173 20 5688 UP 216088_s_at NM_002792; 398; 399 NP_002783; 1149;1150 NM_152255 NP_689468 20 9885 UP 209222_s_at NM_014835; 400; 401NP_055650; 1151; 1152 NM_144498 NP_653081 20 3911 UP 210150_s_atNM_005560 402 NP_005551 1153 20 6227 UP 200834_s_at NM_001024 403NP_001015 1154 20 10732 UP 204849_at NM_006602 404 NP_006593 1155 2054994 UP 218448_at NM_017896 405 NP_060366 1156 20 54915 UP 221741_s_atNM_017798 406 NP_060268 1157 20 10139 UP 215984_s_at NM_003224 407NP_003215 1158 20 84619 UP 221848_at NM_032527; 408; 409; 410 NP_115916;1159; 1160; 1161 NM_181484; NP_852149; NM_181485 NP_852130 20 10139 UP203174_s_at NM_003224 411 NP_003215 1162 20 84619 UP 57539_at NM_032527;412; 413; 414 NP_115916; 1163; 1164; 1165 NM_181484; NP_852149;NM_181485 NP_852150 20 7165 UP 201379_s_at NM_003288; 415; 416; 417;418; NP_003279; 1166; 1167; 1168; NM_199359; 419; 420 NP_955391; 1169;1170; 1171 NM_199360; NP_955392; NM_199361; NP_955393; NM_199362;NP_955394; NM_199363; NP_955395 20 54963 UP 218533_s_at NM_017859 421NP_060329 1172 20 24148 UP 208879_x_at NM_012469 422 NP_036601 1173 221652 UP 202929_s_at NM_001355 423 NP_001346 1174 22 2952 UP 203815_atNM_000853 424 NP_000844 1175 22 55000 UP 222244_s_at NM_017903 425NP_060373 1176 22 55000 UP 212337_at NM_017903 426 NP_060373 1177

What is claimed is:
 1. A method of assessing a subject at risk fordeveloping pancreatic adenocarcinoma, the method comprising: a)obtaining a pancreatic tissue or pancreatic juice sample from a subjectsuspected of being at risk for developing pancreatic adenocarcinomacancer; b) determining a copy number of minimal common region (MCR)50.06-62.89 Mb of human chromosome 19 in the subject pancreatic tissueor pancreatic juice sample using fluorescent in situ hybridization(FISH), quantitative PCR (qPCR), or comparative genomic hybridization(CGH); c) comparing the copy number of the MCR in the subject sample tothe normal diploid copy number of the MCR, wherein an increased copynumber of the MCR in the subject sample indicates that the subject is atrisk for developing pancreatic adenocarcinoma cancer; and d)recommending pancreatic adenocarcinoma treatment to the subject at riskfor developing pancreatic adenocarcinoma.
 2. The method of claim 1,wherein the normal copy number is obtained from a control sample.
 3. Themethod of claim 2, wherein said CGH is performed on an array.